KR20180105730A - Modulation of dystrophia myotonica-protein kinase (dmpk) expression - Google Patents

Abstract

Methods, compounds and compositions for reducing the expression of DMPK mRNA and protein in an animal are provided herein. Also provided herein are methods, compounds, and compositions for selectively reducing CUGexp DMPK RNA in an animal, reducing muscle tension, or reducing splice disruption. Such methods, compounds, and compositions are useful for treating, preventing, delaying, or ameliorating Type I muscle tonic dystrophy or symptoms thereof.

Description

Method for regulating the expression of muscle tone dystrophy-protein kinase (DMPK) {MODULATION OF DYSTROPHIA MYOTONICA-PROTEIN KINASE (DMPK) EXPRESSION}

Sequence list

This application is filed with a sequence listing in electronic format. The sequence list is provided as a file titled BIOL0134USL2SEQ.txt generated on July 19, 2011 with a size of about 216Mb. Information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

Field

Provided herein are methods, compounds and compositions for reducing the expression of DMPK mRNA and proteins in animals. In addition, methods, compounds, and compositions comprising DMPK inhibitors for selectively reducing CUGexp DMPK RNA, reducing muscle tone, or reducing spliceopathy in animals are provided herein. do. These methods, compounds and compositions are useful, for example, to treat, prevent or ameliorate type 1 myotonic dystrophy (DM1) in animals.

Muscular dystrophy type 1 (DM1) is the most common form of muscular dystrophy present in adults with an estimated frequency of 1 in 7,500 (Harper PS., Myotonic Dystrophy. London: WB Saunders Company; 2001). DM1 is an autosomal dominant disease caused by the expansion of non-coding CTG repeats in DMPK1. DMPK1 is a gene encoding cytoplasmic serine/threonine kinase (Brook JD, et al., Cell , 1992 , 68(4) :799-808). The physiological function and substrate of the kinase have not been completely determined. The expanded CTG repeat is located in the 3'untranslated region (UTR) of DMPK1. These mutations cause RNA dominance, a process in which the expression of RNA containing extended CUG repeats (CUGexp) induces cell dysfunction (Osborne RJ and Thornton CA., Human Molecular Genetics. , 2006 , 15(2)) : R162-R169).

The DMPK gene usually has 5-37 CTG repeats within the 3'untranslated region. In myosonic dystrophy type I, this number expands significantly, which ranges, for example, from 50 to more than 3,500 (Harper, Myotonic Dystrophy (Saunders, London, ed. 3, 2001); Annu. Rev. Neurosci. 29: 259, 2006; EMBO J. 19: 4439, 2000; Curr Opin Neurol. 20: 572, 2007).

The CUGexp tract interacts with RNA binding proteins, including the splicing factor muscleblind-like (MBNL) protein, and allows mutant transcripts to remain within the nuclear foci. This RNA toxicity results from the isolation of RNA binding proteins and activation of signaling pathways. Studies in animal models have shown that the phenotype of DM1 can be reversed when the toxicity of CUGexp RNA is reduced (Wheeler TM, et al., Science ., 2009 , 325 (5938):336-339; Mulders SA. , et al., Proc Natl Acad Sci USA ., 2009 , 106 (33):13915-13920).

In DM1, skeletal muscle is the most severely affected tissue, but the disease also has a significant effect on the heart and smooth muscle, ocular lens and brain. The cranial, distal limb and diaphragm muscles are primarily affected. Manual dexterity is initially impaired, causing severe disability for decades. The median age at death is 55 years, and these deaths usually result from respiratory failure (de Die-Smulders CE, et al., Brain ., 1998 , 121 (Pt 8): 1557-1563).

Antisense technology has recently been created as an effective means for regulating the expression of certain gene products, and thus it can be found that it is uniquely useful in a number of therapeutic, diagnostic and research applications for the regulation of DMPK1. Intramuscular injection of a fully modified oligonucleotide targeting the CAG-repeat blocks the formation of the CUGexp-MBNL1 complex in mice, disperses the nuclear foci of CUGexp transcripts, and facilitates nuclear cytoplasmic transport and translation of CUGexp transcripts. It has been shown to enhance, release MBNL protein into the nucleus, normalize alternative splicing of MBNL-dependent exons, and eliminate dystonia in CUGexp-expressing transgenic mice (Wheeler TM, et al., Science . , 2009 , 325 (5938):336-339; WO2008/036406).

Currently, there is no treatment that can modulate the process of DM1. Therefore, the burden of disease is remarkable. Accordingly, it is an object of the present application to provide compounds, compositions and methods for treating DM1.

Provided herein are methods, compounds and compositions for inhibiting the expression of DMPK and treating, preventing, delaying, or ameliorating DMPK-related diseases and/or symptoms thereof. In certain embodiments, the compounds and compositions inhibit mutant DMPK or CUGexp DMPK.

Certain embodiments provide a method of reducing DMPK expression in an animal comprising administering to the animal a compound comprising a modified oligonucleotide as further described herein targeting DMPK.

Certain embodiments selectively reduce CUGexp DMPK or reduce myotonia in an animal comprising administering to the animal a compound comprising a modified oligonucleotide as further described herein targeting CUGexp DMPK. Or, it provides a method of reducing splice error. CUGexp DMPK transcripts are thought to be particularly sensitive to antisense knockdown via nuclear ribonuclease due to their longer residence time in the nucleus, despite the biodistribution barrier to tissue uptake of antisense oligonucleotides. It is believed to enable effective antisense inhibition of CUGexp DMPK transcripts in related tissues such as muscle. For example, cleavage through nuclear ribonuclease such as CAG-repeat ASO described in Wheeler TM, et al., Science., 2009, 325(5938):336-339 and WO2008/036406 Antisense mechanisms that do not induce do not provide the same therapeutic benefits.

Certain embodiments provide a method of treating an animal with type 1 myotonic dystrophy. In certain embodiments, the method comprises administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide as further described herein targeting DMPK. In certain embodiments, the method comprises identifying an animal with type 1 muscular dystrophy.

Specific embodiments include muscle stiffness, muscle tone, distal weakness, weakness of the facial and jaw muscles, difficulty swallowing, weakness in the eyelids (ptosis), weakness of the neck muscles, arms and legs. Treat symptoms and consequences associated with the development of DM1, including muscle weakness, persistent muscle pain, excessive sleep, muscle wasting, dysphagia, respiratory failure, irregular heartbeat, heart muscle damage, numbness, insulin resistance, and cataracts, or It provides a method of preventing, delaying, or improving. Certain embodiments provide methods of treating, preventing, delaying, or ameliorating the symptoms and outcomes associated with the development of DM1 in children, including developmental delays, learning disabilities, language and speech problems, and personality development problems. .

Certain embodiments provide methods of administering antisense oligonucleotides that neutralize RNA dominance by inducing cleavage of pathogenic transcripts.

In certain embodiments, the DMPK has a sequence as listed in GenBank Accession No. NM_001081560.1 (incorporated herein as SEQ ID NO: 1). In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NT_011109.15 (incorporated herein as SEQ ID NO: 2) truncate from nucleotides 18540696 to 18555106. In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NT_039413.7 (incorporated herein as SEQ ID NO: 3) truncate from nucleotides 16666001 to 16681000. In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NM_032418.1 (incorporated herein as SEQ ID NO: 4). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number AI007148.1 (incorporated herein as SEQ ID NO: 5). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number AI304033.1 (incorporated herein as SEQ ID NO: 6). In certain embodiments, the DMPK has a sequence as listed herein under Genbank Accession No. BC024150.1 (incorporated herein as SEQ ID No. 7). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number BC056615.1 (incorporated herein as SEQ ID NO: 8). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number BC075715.1 (incorporated herein as SEQ ID NO: 793). In certain embodiments, the DMPK has a sequence as listed in Genbank accession number BU519245.1 (incorporated herein as SEQ ID NO: 794). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number CB247909.1 (incorporated herein as SEQ ID NO: 795). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession No. CX208906.1 (incorporated herein as SEQ ID No. 796). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number CX732022.1 (incorporated herein as SEQ ID NO: 797). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number S60315.1 (incorporated herein as SEQ ID NO: 798). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number S60316.1 (incorporated herein as SEQ ID NO: 799). In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NM_001081562.1 (incorporated herein as SEQ ID NO: 800). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number NM_001100.3 (incorporated herein as SEQ ID NO: 801).

It is to be understood that both the above general description and the following detailed description are illustrative and illustrative only, and are not intended to limit the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Moreover, the use of the term “comprising” as well as other forms such as “comprises” and “included” is not limited. In addition, terms such as "component" or "component" include both components and components comprising one unit and components and components comprising one or more subunits, unless specifically stated otherwise.

Section headings as used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application in this application, including but not limited to patents, patent applications, literature, books and papers, are by reference in their entirety as well as portions of these documents discussed herein. Specifically included herein.

Justice

Unless a specific definition is provided, the nomenclature used in connection with the procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications and other applications referred to throughout this disclosure, Genbank registration numbers and related sequence information and other data available through databases such as the National Center for Biological Information (NCBI). Is incorporated herein by reference in its entirety, as well as some of these documents discussed herein.

Unless otherwise indicated, the following terms have the following meanings:

"2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH ₂ ) ₂ -OCH ₃ ) refers to the O-methoxy-ethyl modification of the 2'position of the furanosyl ring. . The 2'-O-methoxyethyl modified sugar is a modified sugar.

"2'-O-methoxyethyl nucleotide" means a nucleotide comprising a 2'-O-methoxyethyl modified sugar moiety.

"5-methylcytosine" means a cytosine modified with a methyl group attached to position 5. 5-methylcytosine is a modified nucleobase.

“About” means within ±7% of a value. For example, if it is stated that "the compound affected at least 70% inhibition of DMPK", this means that DMPK levels are inhibited within the range of 63% and 77%.

“Active pharmaceutical agent” means a substance or substances in a pharmaceutical composition that provides a therapeutic benefit when administered to a subject. For example, in certain embodiments, antisense oligonucleotides targeting DMPK are active pharmaceutical agents.

"Active target region" or "target region" means a region to which one or more active antisense compounds are targeted. “Active antisense compound” means an antisense compound that reduces the level of a target nucleic acid or protein.

“Concurrently administered” means the co-administration of two agents in any manner in which the pharmacological effect of the two agents is simultaneously manifested in a patient. Simultaneous administration does not require both agents to be administered in a single pharmaceutical composition, the same dosage form, or the same route of administration. The effect of both agents does not require that the effects of both agents themselves appear at the same time. The effect only needs to be superimposed over a period of time and does not need to coexist.

“Administering” means providing an agent to the animal, including, but not limited to, administration and self-administration by a medical professional.

“Agent” means an active substance capable of providing a therapeutic benefit when administered to an animal. "First agent" means a therapeutic compound of the present invention. For example, the first agent may be an antisense oligonucleotide targeting DMPK. "Second agent" means a second therapeutic compound of the invention (eg, a second antisense oligonucleotide targeting DMPK) and/or a non-DMPK therapeutic compound.

“Improvement” means a reduction in at least one indicator, sign or symptom of a related disease, disorder or condition. The severity of an indicator can be determined by subjective or objective measures known to those skilled in the art.

“Animal” means human or non-human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates including, but not limited to, monkeys and chimpanzees. .

“Antisense activity” means any detectable or measurable activity resulting from the hybridization of an antisense compound to a target nucleic acid of the antisense compound. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or a protein encoded by the target nucleic acid.

"Antisense compound" means an oligomeric compound capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds such as antisense oligonucleotides, siRNA, shRNA, snoRNA, miRNA and satellite repeats.

“Antisense inhibition” refers to a reduction in the level of a target nucleic acid or a level of a target protein in the presence of an antisense compound complementary to the target nucleic acid compared to the level of the target nucleic acid or the level of the target protein in the absence of the antisense compound.

“Antisense oligonucleotide” means a single stranded oligonucleotide having a nucleobase sequence that allows hybridization to a corresponding region or segment of a target nucleic acid.

"Bicyclic sugar" means a furanosyl ring modified by bridging two non-geminal carbon ring atoms. Bicyclic sugars are modified sugars.

"Bicyclic nucleic acid" or "BNA" refers to a nucleoside or nucleotide that forms a bicyclic ring system by including a bridge in which the furanose portion of the nucleoside or nucleotide comprises a bridge connecting two carbon atoms on the furanose ring. it means.

“Cap structure” or “terminal cap moiety” refers to a chemical modification incorporated at either end of an antisense compound.

"Chemically distinct regions" means regions of an antisense compound that are in some way chemically different from another region of the same antisense compound. For example, a region having a 2'-O-methoxyethyl nucleotide is chemically distinct from a region having a nucleotide that does not have a 2'-O-methoxyethyl modification.

“Chimeric antisense compound” means an antisense compound having at least two chemically distinct regions.

“Co-administration” means administration of two or more agents to a subject. Two or more agents can exist in a single pharmaceutical composition, or can exist in separate pharmaceutical compositions. Each of the two or more agents may be administered via the same or different routes of administration. Co-administration includes concurrent or sequential administration.

"Complementary" means the ability to form a pair between the nucleobase of a first nucleic acid and a second nucleic acid.

"Continuous nucleobases" means nucleobases immediately adjacent to each other.

“CUGexp DMPK” means a mutant DMPK RNA containing extended CUG repeats (CUGexp). The wild-type DMPK gene has 5-37 CTG repeats in the 3'untranslated region. In “CUGexp DMPK” (eg, in myosonic dystrophy type I patients), this number expands significantly, which ranges, for example, from 50 to more than 3,500 (Harper, Myotonic Dystrophy (Saunders, London , ed. 3, 2001); Annu. Rev. Neurosci. 29: 259, 2006; EMBO J. 19: 4439, 2000; Curr Opin Neurol. 20: 572, 2007).

"Diluent" refers to an ingredient in a composition that is lacking in pharmacological activity, but is pharmaceutically necessary or desired. For example, the component in the composition to be injected may be a liquid, such as a saline solution.

“DMPK” means any nucleic acid or protein of DMPK. The DMPK may be a mutant DMPK comprising a CUGexp DMPK nucleic acid.

"DMPK expression" means the level of mRNA transcribed from a gene encoding DMPK or the level of protein translated from the mRNA. DMPK expression can be determined by methods known in the art such as Northern or Western blot.

“DMPK nucleic acid” means any nucleic acid encoding DMPK. For example, in certain embodiments, the DMPK nucleic acid is a DNA sequence encoding DMPK, an RNA sequence transcribed from DNA encoding DMPK (including genomic DNA including introns and exons), and mRNA encoding DMPK or Contains pre-mRNA sequences. "DMPK mRNA" means an mRNA encoding a DMPK protein.

"Dose" means a single dose or a specific amount of a pharmaceutical agent given over a specific period of time. In certain embodiments, the dose may be administered as one, two or more boluses, tablets or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that is not readily provided by a single injection, so two or more injections may be used to achieve the desired dose. . In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Dosage can be referred to as the amount of the pharmaceutical agent per hour, day, week or month.

“Effective amount” or “therapeutically effective amount” means an amount of an active pharmaceutical agent sufficient to achieve the desired physiological result in an individual in need of the agent. The effective amount may vary from individual to individual depending on the health and physical condition of the individual being treated, the classification of the individual being treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

“Fully complementary” or “100% complementary” means that each nucleobase of the nucleobase sequence of the first nucleic acid has a complementary nucleobase within the second nucleobase sequence of the second nucleic acid. In certain embodiments, the first nucleic acid is an antisense compound and the target nucleic acid is a second nucleic acid.

"Gapmer" refers to a chimeric antisense compound in which an inner region having a plurality of nucleosides supporting RNase H cleavage is located between an outer region having one or more nucleosides, and a nucleoside comprising the inner region is It is chemically distinct from the nucleosides or nucleosides comprising the outer region. The inner region may be referred to as a “gap segment” and the outer region may be referred to as a “wing segment”.

"Gap-expanded" is a chimera having a gap segment of 12 or more consecutive 2'-deoxyribonucleosides located between 5'and 3'wing segments having 1 to 6 nucleosides and immediately adjacent thereto. It means an antisense compound.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, the complementary nucleic acid molecule comprises an antisense compound and a target nucleic acid.

"Identifying an animal with type 1 muscular dystrophy" means identifying an animal diagnosed as having a 1 muscular dystrophy disorder or disease, or identifying an animal prone to develop a type 1 muscular dystrophy disorder or disease. For example, an individual with a family history may be susceptible to developing a type 1 muscular dystrophy disorder or disease. Such identification can be achieved by any method including evaluating the individual's medical history and standard clinical trials or evaluations.

"Immediately adjacent" means that there are no intervening components in the immediate vicinity between the components.

“Individual” means a human or non-human animal selected for treatment or therapy.

"Internucleoside bond" means a chemical bond between nucleosides.

"Bound nucleosides" means adjacent nucleosides that are joined or linked together by nucleoside bonds.

"Mismatch" or "non-complementary nucleobase" means when the nucleobase of the first nucleic acid is unable to pair with the corresponding nucleobase of the second or target nucleic acid.

"Modified internucleoside linkage" means a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).

"Modified nucleobase" means any nucleobase that is not adenine, cytosine, guanine, thymidine or uracil. “Unmodified nucleobase” means purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).

"Modified nucleotide" means a nucleotide that independently has a modified sugar moiety, a modified internucleoside bond or a modified nucleobase. “Modified nucleoside” means a nucleoside that independently has a modified sugar moiety or a modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleotide.

"Modified sugar" means a substitution or modification from a natural sugar.

“Motif” refers to a pattern of chemically distinct regions within an antisense compound.

"Myotonia" means abnormal slow relaxation of a muscle after voluntary contraction or electrical stimulation.

"Nuclear ribonuclease" means a ribonuclease found in the nucleus. Nuclear ribonucleases include, but are not limited to, RNase H, including RNase H1 and RNase H2, double-stranded RNase drosha, and other double-stranded RNases.

"Naturally occurring internucleoside linkage" means a phosphodiester linkage from 3'to 5'.

“Natural sugar moiety” means a sugar found in DNA (2'-H) or RNA (2'-OH).

"Nucleic acid" means a molecule composed of monomeric nucleotides. Nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA) and microRNA (miRNA). Nucleic acids may also contain a combination of these components within a single molecule.

"Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

"Nucleobase sequence" means a sequence of consecutive nucleobases independent of any sugar, bond or nucleobase modification.

"Nucleoside" means a nucleobase bound to a sugar.

“Nucleoside mimetic” refers to, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, such as non-furanose sugar units A sugar or sugar and a base at one or more positions of an oligomeric compound, such as a nucleoside mimetic having, and not necessarily, a structure used to replace a bond.

"Nucleoside" means a nucleoside having a phosphate group covalently bonded to the sugar moiety of the nucleoside.

A “nucleotide mimic” is, for example, a peptide nucleic acid or morpholino (morpholino is linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Includes structures used to replace nucleosides and bonds at one or more positions of an oligomeric compound.

“Oligomer compound” or “oligomer” means a polymer of bound monomeric subunits capable of hybridizing to at least one region of a nucleic acid molecule.

"Oligonucleotide" means a polymer of linked nucleosides, each of which may or may not be modified independently of one another.

“Parental administration” means administration via injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intraventricular administration. Administration may be continuous, or chronic, or short, or intermittent.

“Peptide” refers to a molecule formed by linking at least two amino acids by amino bonds. Peptide refers to polypeptides and proteins.

"Pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, the pharmaceutical composition may comprise one or more active agents and a sterile aqueous solution.

“Pharmaceutically acceptable salt” is a physiological and pharmaceutically acceptable salt of an antisense compound, that is, retains the desired biological activity of the parent oligonucleotide, and has an undesirable toxicological effect on the parent oligonucleotide. It means a salt that does not provide.

“Phosphorothioate bond” means a bond between nucleosides wherein a phosphodiester bond is modified by replacing one of the oxygen atoms that do not form a bridge with a sulfur atom. Phosphorothioate bonds are modified internucleoside bonds.

"Part" means a defined number of contiguous (ie, bound) nucleobases of a nucleic acid. In certain embodiments, the portion is a defined number of consecutive nucleobases of the target nucleic acid. In certain embodiments, the moiety is a defined number of consecutive nucleobases of the antisense compound.

“Selectively reducing CUG exp DMPK RNA” refers to a selective reduction of RNA transcripts from the CUGexp DMPK allele compared to RNA transcripts from normal DMPK alleles.

"Prevent" means to delay or prevent the occurrence or development of a disease, disorder or condition for a period of minutes to indefinitely. Prevention also means that the disease, disorder or condition reduces the risk of development.

"Prodrug" means a therapeutic agent that is prepared in an inactive form that is converted into an active form in the body or in cells by the action of an endogenous enzyme or other compound or condition.

"Side effect" refers to a physiological response resulting from treatment that is not the desired effect. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, kidney function abnormalities, liver toxicity, kidney toxicity, central nervous system abnormalities, myopathy and malaise. For example, elevated aminotransferase levels in serum can indicate liver toxicity or liver dysfunction. For example, increased bilirubin can indicate liver toxicity or liver dysfunction.

“Single stranded oligonucleotide” means an oligonucleotide that has not hybridized to a complementary strand.

“Specifically hybridizable” means a degree sufficient between the antisense oligonucleotide and the target nucleic acid to induce the desired effect under conditions in which specific binding is desired, ie, in the case of in vivo assays and therapeutic treatments, under physiological conditions. It has the complementarity of, but exhibits minimal or no effect on non-target nucleic acid means an antisense compound.

“Splice opacity” means a change in alternative splicing of one or more RNAs that results in the expression of an altered splice product within a particular tissue.

“Subcutaneous administration” means administration just below the skin.

“Sugar surrogate” overlaps with the slightly broader term “nucleoside mimetic”, but is intended to denote the replacement of only sugar units (furanose rings). The tetrahydropyranyl ring provided herein exemplifies an example of a sugar substitute in which a furanose sugar group has been replaced with a tetrahydropyranyl ring system.

“Targeting” or “targeted” refers to the process of designing and selecting antisense compounds that specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid”, “target RNA” and “target RNA transcript” all refer to a nucleic acid that can be targeted by an antisense compound.

“Target segment” means the sequence of nucleotides of a target nucleic acid targeted by an antisense compound. "5' target site" means the 5'most nucleotide of a target segment. "3' target site" means the 3'most nucleotide of a target segment.

"Therapeutically effective amount" means an amount of an agent that provides a therapeutic benefit to an individual.

"Treat" means administering a pharmaceutical composition to achieve alteration or amelioration of a disease, disorder or condition.

"Type 1 muscular dystrophy" or "DM1" refers to an autosomal dominant disorder caused by the expansion of non-coding CTG repeats in DMPK. These mutations give rise to RNA dominance, a process by which the expression of RNA containing extended CUG repeats (CUGexp) induces cellular dysfunction. The CUGexp track interacts with the RNA binding protein and allows mutant transcripts to remain within the nuclear foci. This RNA toxicity results from the isolation of RNA binding proteins and activation of signaling pathways.

“Unmodified nucleotide” means a nucleotide consisting of a naturally occurring nucleobase, a sugar moiety, and an internucleoside linkage. In certain embodiments, the unmodified nucleotide is an RNA nucleotide (ie, β-D-ribonucleoside) or a DNA nucleotide (ie, β-D-deoxyribonucleoside).

Specific embodiment

Certain embodiments provide methods, compounds and compositions for inhibiting DMPK expression.

Certain embodiments provide a method of reducing DMPK expression in an animal comprising administering to the animal a compound comprising a modified oligonucleotide targeting DMPK.

Certain embodiments selectively reduce CUGexp DMPK RNA, reduce muscle tone, or reduce spliceopathy in an animal comprising administering to the animal a compound comprising a modified oligonucleotide targeting DMPK. And the modified oligonucleotide selectively reduces CUGexp DMPK RNA, reduces myotonia, or reduces spliceopathy in animals.

Certain embodiments provide methods of administering antisense oligonucleotides that neutralize RNA dominance by inducing cleavage of pathogenic transcripts.

Certain embodiments provide a method of reducing spliceopacity of Serca1. In certain embodiments, the methods provided herein result in exon 22 inclusion. In certain embodiments, orthodontic splicing occurs in the anterior tibialis, calf muscles and quadriceps muscles.

Certain embodiments provide a method of reducing spliceopacity of m-Titin. In certain embodiments, the methods provided herein result in exon 5 encapsulation. In certain embodiments, orthodontic splicing occurs in the anterior tibialis, calf muscles and quadriceps muscles.

Certain embodiments provide a method of reducing the spliceopacity of Clcn1. In certain embodiments, the methods provided herein result in exon 7a encapsulation. In certain embodiments, orthodontic splicing occurs in the anterior tibialis, calf muscles and quadriceps muscles.

Certain embodiments provide a method of reducing spliceopacity of Zasp. In certain embodiments, the methods provided herein result in exon 11 encapsulation. In certain embodiments, orthodontic splicing occurs in the anterior tibialis, calf muscles and quadriceps muscles.

Certain embodiments comprise the steps of: a) selecting an animal with type 1 muscular dystrophy, and b) administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide targeting DMPK. , Provides a method of treating an animal with type 1 dystrophic dystrophy. In certain embodiments, a therapeutically effective amount of a compound administered to an animal selectively reduces CUGexp DMPK RNA, reduces myotonia, or reduces spliceopacity in the animal.

A specific embodiment is a CUGexp DMPK RNA comprising the step of administering a modified antisense oligonucleotide complementary to the non-repeating region of the CUGexp DMPK RNA to a subject suspected of having type 1 dystrophy or CUGexp DMPK RNA. Provides a way to achieve selective reduction. The modified antisense oligonucleotide achieves a selective reduction of CUGexp DMPK RNA when bound to the CUGexp DMPK RNA.

A specific embodiment comprises the steps of selecting a subject having type 1 dystrophy or CUGexp DMPK RNA, and administering to the subject a modified antisense oligonucleotide complementary to the non-repeated region of the CUGexp DMPK RNA. It provides a method of achieving the selective reduction of containing CUGexp DMPK RNA. Modified antisense oligonucleotides achieve selective reduction of CUGexp DMPK RNA in the nucleus by activating ribonuclease or nuclear ribonuclease when binding to the CUGexp DMPK RNA.

A specific embodiment is the step of selecting a subject having type 1 dystrophy or mutation or CUGexp DMPK RNA, and systemically administering a modified antisense oligonucleotide complementary to the non-repeating region of the CUGexp DMPK RNA to the subject. It provides a method of achieving the selective reduction of CUGexp DMPK RNA comprising the step of. The modified antisense oligonucleotide achieves the mutation or selective reduction of CUGexp DMPK RNA when bound to the mutation or CUGexp DMPK RNA.

Certain embodiments provide a method of reducing myotonia in a subject in need thereof. The method comprises administering to a subject a modified antisense oligonucleotide complementary to a non-repeating region of DMPK RNA, wherein the modified antisense oligonucleotide is ribonuclease or nuclear ribonucleotide when bound to DMPK RNA. Reduces muscle tone by activating nuclease. In certain embodiments, the subject has, or is suspected of having, type 1 myotonic dystrophy or mutant DMPK RNA or CUGexp DMPK RNA. In certain embodiments, the DMPK RNA is nuclear retained.

Certain embodiments provide a method of reducing splice errors in a subject in need of reducing splice errors. The method comprises administering to a subject a modified antisense oligonucleotide complementary to a non-repeating region of DMPK RNA, wherein the modified antisense oligonucleotide is ribonuclease or nuclear ribonucleotide when bound to DMPK RNA. By activating nucleases, splice opacity is reduced. In certain embodiments, the subject has, or is suspected of having, type 1 myotonic dystrophy or nuclear retained CUGexp DMPK RNA. In certain embodiments, the DMPK RNA is nuclear retained. In a specific embodiment, the spliceofface is an MBNL dependent spliceofface.

In certain embodiments, the modified antisense oligonucleotide of the method is a chimeric antisense oligonucleotide. In certain embodiments, the modified antisense oligonucleotide of the method is a gapmer.

In certain embodiments of the methods provided herein, the administration is subcutaneous administration. In certain embodiments, administration is intravenous.

In certain embodiments, the modified antisense oligonucleotide of the method targets a non-coding sequence within the non-repeat region of DMPK RNA. In certain embodiments, the oligonucleotide targets the coding region, intron, 5'UTR or 3'UTR of the mutant DMPK RNA.

In certain embodiments of the methods provided herein, the nuclear ribonuclease is RNase H1.

In certain embodiments of the method, DMPK RNA is reduced in muscle tissue. In certain embodiments, the mutant DMPK RNA CUGexp DMPK RNA is selectively reduced.

In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NM_001081560.1 (incorporated herein as SEQ ID NO: 1). In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NT_011109.15 (incorporated herein as SEQ ID NO: 2) truncate from nucleotides 18540696 to 18555106. In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NT_039413.7 (incorporated herein as SEQ ID NO: 3) truncate from nucleotides 16666001 to 16681000. In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NM_032418.1 (incorporated herein as SEQ ID NO: 4). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number AI007148.1 (incorporated herein as SEQ ID NO: 5). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number AI304033.1 (incorporated herein as SEQ ID NO: 6). In certain embodiments, the DMPK has a sequence as listed herein under Genbank Accession No. BC024150.1 (incorporated herein as SEQ ID No. 7). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number BC056615.1 (incorporated herein as SEQ ID NO: 8). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number BC075715.1 (incorporated herein as SEQ ID NO: 793). In certain embodiments, the DMPK has a sequence as listed in Genbank accession number BU519245.1 (incorporated herein as SEQ ID NO: 794). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number CB247909.1 (incorporated herein as SEQ ID NO: 795). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession No. CX208906.1 (incorporated herein as SEQ ID No. 796). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number CX732022.1 (incorporated herein as SEQ ID NO: 797). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number S60315.1 (incorporated herein as SEQ ID NO: 798). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number S60316.1 (incorporated herein as SEQ ID NO: 799). In certain embodiments, the DMPK has a sequence as listed in Genbank accession number NM_001081562.1 (incorporated herein as SEQ ID NO: 800). In certain embodiments, the DMPK has a sequence as listed in Genbank Accession Number NM_001100.3 (incorporated herein as SEQ ID NO: 801).

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 8 contiguous nucleobases of the nucleobase sequences listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. In certain embodiments, the modified oligonucleotide comprises at least 9, at least 10, or at least 11 consecutive nucleobases of the nucleobase sequence listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. It has a nucleobase sequence.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 12 contiguous nucleobases of the nucleobase sequences listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence comprising at least 13 or at least 14 consecutive nucleobases of the nucleobase sequence listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. Have.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 15 contiguous nucleobases of the nucleobase sequences listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence comprising at least 16 or at least 17 contiguous nucleobases of the nucleobase sequence listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. Have.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 18 contiguous nucleobases of the nucleobase sequences listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 19 contiguous nucleobases of the nucleobase sequences listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792.

In certain embodiments, the modified oligonucleotides provided herein comprise any of the regions of SEQ ID NOs: 1:1178-1206, 2159-2182, 2174-2196, 2426-2447, 2450-2518, 2679-2704 and 2697-2725. Target.

In certain embodiments, the modified oligonucleotides provided herein are SEQ ID NOs: 1:178-223, 232-253, 279-299, 366-399, 519-541, 923-975, 1073-1105, 1171-1196, 1215 -Targets any of the areas of 1246, 1263-1324, 1706-1734, 1743-1763, 1932-1979, 1981-2003, 2077-2108 and 2152-2173.

In certain embodiments, the modified oligonucleotides provided herein are SEQ ID NOs: 2:1251-1303, 1305-1326, 1352-1372, 3762-3795, 4170-4192, 5800-5852, 6124-6149, 6168-6199, 6216 It targets any of the areas of -6277, 11979-12007, 12016-12036, 12993-13042, 13044-13066, 13140-13171 and 13215-13236.

In certain embodiments, the animal is human.

In certain embodiments, the compound or composition of the invention is designated as a first agent, and the methods of the invention further comprise administering a second agent. In certain embodiments, the first agent and the second agent are administered co-administered. In certain embodiments, the first agent and the second agent are administered sequentially or concurrently.

In certain embodiments, administration includes parenteral administration.

In certain embodiments, the compound is a single stranded modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to any one of SEQ ID NOs: 1-8 and 793-801 as measured throughout the modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to any one of SEQ ID NOs: 1-8 and 793-801 as measured throughout the modified oligonucleotide.

In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, the at least one modified sugar is a bicyclic sugar. In certain embodiments, the at least one modified sugar comprises 2'-0-methoxyethyl, or a 4'-(CH ₂ ) _n -O-2' bridge in which n is 1 or 2.

In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises a) a gap segment consisting of bound deoxynucleosides; b) a 5'wing segment consisting of linked nucleosides; And c) a 3'wing segment consisting of linked nucleosides. The gap segment is located between the 5'wing segment and the 3'wing segment, and each nucleoside of each wing segment contains a modified sugar.

In certain embodiments, the modified oligonucleotide comprises a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5'wing segment consisting of 5 linked nucleosides; And c) a 3'wing segment consisting of 5 linked nucleosides. The gap segment is located between the 5'wing segment and the 3'wing segment, and each nucleoside of each wing segment contains a 2'-0-methoxyethyl sugar, and each nucleus of the modified oligonucleotide. The cleoside bond is a phosphorothioate bond, and each cytosine in the modified oligonucleotide is 5'-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.

Certain embodiments have a gap segment consisting of a 5'wing segment consisting of 10 linked deoxynucleosides, a 5'wing segment consisting of 5 linked nucleosides, and a 3'wing segment consisting of 5 linked nucleosides. It provides a method of selectively reducing CUGexp DMPK RNA, reducing myotonia, or reducing spliceopacity in an animal comprising administering to the animal a compound comprising a modified oligonucleotide. The gap segment is located between the 5'wing segment and the 3'wing segment, and each nucleoside of each wing segment contains a 2'-0-methoxyethyl sugar, and each nucleus of the modified oligonucleotide. The cleoside bond is a phosphorothioate bond, and each cytosine in the modified oligonucleotide is 5'-methylcytosine.

Certain embodiments provide for the use of any compound as described herein in the manufacture of a medicament for use in any of the methods of treatment described herein. For example, certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for treating, ameliorating, or preventing type 1 muscular dystrophy. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for inhibiting the expression of DMPK and treating, preventing, delaying, or ameliorating DMPK related diseases and/or symptoms thereof. Certain embodiments provide for the use of a compound as described herein in the manufacture of a medicament for reducing DMPK expression in an animal. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for selectively reducing CUGexp DMPK, reducing myotonia, or reducing spliceopathy in an animal. Certain embodiments provide for the use of a compound as described herein in the manufacture of a medicament for the treatment of an animal with type 1 muscular dystrophy. Specific embodiments include muscle stiffness, muscle tone, distal weakness, weakness of the facial and jaw muscles, difficulty swallowing, weakness in the eyelids (ptosis), weakness of the neck muscles, arms and legs. Treat or prevent symptoms and consequences associated with the development of DM1, including muscle weakness, persistent muscle pain, excessive sleep, muscle wasting, dysphagia, respiratory failure, irregular heartbeat, heart muscle damage, numbness, insulin resistance, and cataracts. Use of a compound as described herein in the manufacture of a medicament for performing, delaying, or improving is provided. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament that neutralizes RNA dominance by inducing degradation of pathogenic transcripts.

Certain embodiments provide kits for treating, preventing, or ameliorating type 1 muscular dystrophy as described herein, the kit comprising a) a compound as described herein, and optionally b) a compound as described herein. And additional agents or therapy. The kit may further include instructions or labels for using the kit to treat, prevent, or ameliorate type 1 muscular dystrophy.

Certain embodiments provide any compound or composition as described herein for use in any of the methods of treatment described herein. For example, certain embodiments provide a compound or composition as described herein for inhibiting the expression of DMPK and treating, preventing, delaying, or ameliorating DMPK related diseases and/or symptoms thereof. Certain embodiments provide a compound or composition as described herein for use in reducing DMPK expression in an animal. Certain embodiments provide a compound or composition as described herein for use in selectively reducing CUGexp DMPK, reducing myotonia, or reducing spliceopathy in an animal. Certain embodiments provide a compound or composition as described herein for use in treating an animal with type 1 myotonic dystrophy. Certain embodiments include muscle stiffness, dystonia, disability distal weakness, facial and jaw muscles weakness, difficulty swallowing, eyelid weakness (ptosis), neck muscle weakness, arm and leg muscles weakness, persistent muscle pain, Treat, prevent, delay, or ameliorate the symptoms and consequences associated with the development of DM1, including excessive sleep, muscle wasting, dysphagia, respiratory failure, irregular heartbeat, heart muscle damage, numbness, insulin resistance, and cataracts. Compounds or compositions as described herein are provided for use in making. Certain embodiments provide a compound or composition as described herein for use in neutralizing RNA dominance by inducing cleavage of a pathogenic transcript. Certain embodiments include 12 to 30 linked nucleos having a nucleobase sequence comprising at least 12 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-156, 160-770 and 774-792. It provides a compound comprising a modified oligonucleotide consisting of sides.

Other compounds that can be used in the methods described herein are also provided.

For example, certain embodiments include at least 8, at least 9 of any of the nucleobase sequences of SEQ ID NOs: 41, 44, 76, 109, 153, 320, 321, 322, 325, 329, 335 and 657, 10 to 80, 12 to 50, 12 having a nucleobase sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleobases To 30, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleosides.

Certain embodiments include at least 8, at least 9, at least 10, at least of any of the nucleobase sequences of SEQ ID NOs: 15, 73, 77, 79, 83, 85, 130, 602, 648, 655, 674 and 680. 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, 10 to 80, 12 to 50, 12 to 30, 15 having a nucleobase sequence comprising at least 19 consecutive nucleobases To 30, 18 to 24, 19 to 22, or 20 linked nucleosides.

Specific embodiments include the nucleobases 664-683, 773-792, 926-945, 927-946, 928-947, 931-950, 935-954, 941-960, 2089-2108, 2163-2182, At least 8, at least 9, at least 10 complementary to the same length portions of 2490-2509, 2499-2518, 2676-2695, 2685-2704, 2676-2695, 2688-2707, 2697-2716, 2764-2783 and 2770-2789 , 10 to 80, 12 having a nucleobase sequence comprising portions of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 or more contiguous nucleobases. To 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or provides a compound comprising a modified oligonucleotide consisting of 20 linked nucleosides, wherein the nucleobase sequence is SEQ ID NO: 1 Is complementary to

Specific embodiments include the nucleobases 812-831, 3629-3648, 4447-4466, 4613-4632, 5803-5822, 5804-5823, 5805-5824, 5808-5827, 5818-5837, 6794-6813 of SEQ ID NO: 2, At least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 complementary to the same length portion of 12463-12482, 13152-13171 and 13553-13572 Or 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleobases having a nucleobase sequence comprising portions of at least 19 or more contiguous nucleobases. It provides a compound comprising a modified oligonucleotide consisting of nucleosides, wherein the nucleobase sequence is complementary to SEQ ID NO: 2.

In certain embodiments, the modified oligonucleotide is a single stranded oligonucleotide.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of any of SEQ ID NOs: 1-8 and 793-801. Or 100% complementary.

In certain embodiments, the at least one internucleoside linkage is a modified internucleoside linkage.

In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside comprises a modified sugar.

In certain embodiments, the at least one modified sugar is a bicyclic sugar.

In certain embodiments, the at least one modified sugar comprises 2'-0-methoxyethyl.

In certain embodiments, at least one nucleoside comprises a modified nucleobase.

In certain embodiments, the modified nucleobase is 5-methylcytosine.

In certain embodiments, the modified oligonucleotide is

A gap segment consisting of bound deoxynucleosides;

A 5'wing segment consisting of linked nucleosides; And

It includes a 3'wing segment consisting of linked nucleosides,

The gap segment is located between the 5'wing segment and the 3'wing segment, and each nucleoside of each wing segment contains a modified sugar.

In certain embodiments, the modified oligonucleotide is

A gap segment consisting of 10 bound deoxynucleosides;

A 5'wing segment consisting of 5 linked nucleosides; And

It contains a 3'wing segment consisting of 5 linked nucleosides,

The gap segment is located between the 5'wing segment and the 3'wing segment, and each nucleoside of each wing segment contains a 2'-O-methoxyethyl sugar, and each nucleoside bond is It is a phosphorothioate bond.

In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.

Antisense compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleotides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides and siRNA. The oligomeric compound may be “antisense” to the target nucleic acid, meaning that it is capable of undergoing hybridization to the target nucleic acid through hydrogen bonding.

In certain embodiments, the antisense compound has a nucleobase sequence comprising the reverse complement of the target segment of the target nucleic acid to be targeted when described in the 5'to 3'direction. In this specific embodiment, the antisense oligonucleotide has a nucleobase sequence comprising the reverse complement of the target segment of the target nucleic acid to be targeted when described in the 5'to 3'direction.

In certain embodiments, antisense compounds targeting DMPK as described herein are 10 to 30 nucleotides in length. That is, the antisense compound is in some embodiments 10 to 30 linked nucleobases. In other embodiments, the antisense compound is a modified oligonucleotide consisting of 8 to 80, 10 to 80, 12 to 30, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleobases. Includes. In this specific embodiment, the antisense compound is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79 or 80 linked nucleobases in length, or a range defined by any two of the above values. In certain embodiments, the antisense compound of any of the lengths is at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 of the nucleobase sequence of any of the exemplary antisense compounds described herein, At least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleobases (e.g., at least of the nucleobase sequence listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792 8 consecutive nucleobases).

In certain embodiments, the antisense compound comprises a shortened or truncated modified oligonucleotide. A shortened or truncated modified oligonucleotide may have a single nucleoside deleted from the 5'end (5' truncation), or alternatively from the 3'end (3' truncation). Shortened or truncated oligonucleotides can have two nucleosides deleted from the 5'end, or alternatively can have two subunits deleted from the 3'end. Alternatively, the deleted nucleoside is dispersed throughout the modified oligonucleotide in an antisense compound having, for example, one nucleoside deleted from the 5'and one nucleoside deleted from the 3'end. Can be.

When a single additional nucleoside is present in the extended oligonucleotide, the additional nucleoside may be located at the 5'or 3'end of the oligonucleotide. If two or more additional nucleosides are present, the added nucleosides are, for example, added to the 5'end (5' addition) of the oligonucleotide, or alternatively to the 3'end (3' addition). Among oligonucleotides having two nucleosides, they may exist adjacent to each other. Alternatively, the added nucleoside can be dispersed throughout the antisense compound in an oligonucleotide having, for example, one nucleoside added to the 5'end and one subunit added to the 3'end. have.

It is possible to increase or decrease the length of an antisense compound such as an antisense oligonucleotide and/or to introduce a massmatch base without removing activity. See, for example, Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992)], the ability of a series of antisense oligonucleotides of 13-25 nucleobases in length to induce cleavage of target RNA in an oocyte injection model was examined. Was tested. Antisense oligonucleotides with a length of 25 nucleobases with 8 or 11 mismatch bases near the end of the antisense oligonucleotide are to a lesser degree than antisense oligonucleotides without mismatches, but can induce specific cleavage of the target mRNA. there was. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

In Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001), the expression of both bcl-2 and bcl-xL was reduced in bcl-2 mRNA in vitro and in vivo. The ability of oligonucleotides with 100% complementarity to and three mismatches to bcl-xL mRNA was demonstrated. In addition, these oligonucleotides showed potent anti-tumor activity in vivo.

14 nucleobase antisense oligos of a series of tandems for the ability to inhibit the translation of human DHFR in rabbit reticulocyte assay in Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988). Nucleotide, and 28 and 42 nucleobase antisense oligonucleotides consisting of a sequence of 2 or 3 tandem antisense oligonucleotides, respectively, were tested. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation despite a milder level than the 28 or 42 nucleobase antisense oligonucleotides alone.

Antisense compound motif

In certain embodiments, the antisense compound targeting the DMPK nucleic acid confers properties to the antisense compound, such as enhanced inhibitory activity, increased binding affinity for the target nucleic acid, or resistance to degradation by nucleases in vivo. It has chemically modified subunits arranged in a pattern or motif.

Chimeric antisense compounds typically contain at least one region modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid and/or increased inhibitory activity. . The second region of the chimeric antisense compound can optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense compounds with a gapmer motif are considered chimeric antisense compounds. In a gapmer, an inner region with multiple nucleotides supporting RNaseH cleavage is located between the nucleosides of the inner region and an outer region with multiple nucleotides that are chemically different. For antisense oligonucleotides with a gapmer motif, the gap segment generally serves as a substrate for endonuclease cleavage, while the wing segment contains modified nucleosides. In certain embodiments, regions of the gapmer are distinguished by the type of sugar moiety comprising each distinct region. The type of sugar moiety used to distinguish the region of the gapmer is in some embodiments β-D-ribonucleoside, β-D-deoxyribonucleoside, 2'-modified nucleoside (such as 2' -The modified nucleoside may comprise 2'-MOE, especially 2'-O-CH ₃ ), and a modified nucleoside per bicyclic (such a modified nucleoside per bicyclic is 4 It may include those having a'-(CH ₂ ) _n -O-2' bridge, where n=1 or n=2). Preferably, each distinct region contains a homogeneous sugar moiety. The wing-gap-wing motif is often described as "XYZ", where "X" is the length of the 5'wing area, "Y" is the length of the gap area, and "Z" is the length of the 3'wing area. As used herein, a gapmer described as “XYZ” has a shape such that the gap segment is positioned immediately adjacent to each of the 5'wing segment and the 3'wing segment. Thus, there is no nucleotide intervening between the 5'wing segment and the gap segment, or between the gap segment and the 3'wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, and in other embodiments, they are different. In one preferred embodiment, Y is 8 to 15 nucleotides. X, Y or Z are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 Or any of more nucleotides. Thus, gapmers are, for example, 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2 , 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6, 5-8-5, 1-8-1 or 2 Including, but not limited to -6-2.

In certain embodiments, the antisense compound as a “wingmer” motif has a wing-gap or gap-wing form, ie, an X-Y or Y-Z form as described above for a gapmer form. Thus, the wingmer form is, for example, 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13 or 5-13.

In certain embodiments, antisense compounds targeting DMPK nucleic acids have a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeting DMPK nucleic acids have a gap-expanded motif.

In certain embodiments, the antisense compound of any of the gapmer or wingmer motifs is at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 of the nucleobase sequence of any of the exemplary antisense compounds described herein, At least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleobases (e.g., at least one of the nucleobase sequences listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792 8 consecutive nucleobases).

Target nucleic acid, target region and nucleotide sequence

The nucleotide sequence encoding DMPK is, by way of example, non-limiting example, Genbank registration number NM_001081560.1 (incorporated herein as SEQ ID NO: 1), Genbank registration number NT_011109.15 (SEQ ID NO: 2) truncate from nucleotides 18540696 to 18555106 (as SEQ ID NO: 2) Included herein, Genbank registration number NT_039413.7 (incorporated herein as SEQ ID NO: 3), Genbank registration number NM_032418.1 (incorporated herein as SEQ ID NO: 4), Truncate from nucleotide 16666001 to 16681000, Genbank registration number AI007148.1 (incorporated herein as SEQ ID NO: 5), Genbank registration number AI304033.1 (incorporated herein as SEQ ID NO: 6), Genbank registration number BC024150.1 (incorporated herein as SEQ ID NO: 7), Genbank registration No. BC056615.1 (incorporated herein as SEQ ID NO: 8), Genbank registration number BC075715.1 (incorporated herein as SEQ ID NO: 793), Genbank registration number BU519245.1 (incorporated herein as SEQ ID NO: 794), Genbank Registration number CB247909.1 (incorporated herein as SEQ ID NO: 795), Genbank registration number CX208906.1 (incorporated herein as SEQ ID NO: 796), Genbank registration number CX732022.1 (incorporated herein as SEQ ID NO: 797), gene Bank registration number S60315.1 (incorporated herein as SEQ ID NO: 798), Genbank registration number S60316.1 (incorporated herein as SEQ ID NO: 799), Genbank registration number NM_001081562.1 (incorporated herein as SEQ ID NO: 800) and Includes sequences as listed in GenBank Accession No. NM_001100.3 (incorporated herein as SEQ ID NO: 801.) Sequences listed in each of the SEQ ID NOs of the Examples included herein are sugar moieties, nucleoside linkages, or It is understood that it is independent of any modification to the nucleobase. As such, the antisense compounds defined by SEQ ID NOs are independently sugar moieties, nucleoside bonds, or nucleobases. May contain more than one variation. Antisense compounds described by Isis Number (Isis No) represent a combination of nucleobase sequence and motif.

In certain embodiments, the target region is a structurally defined region of a target nucleic acid. For example, the target region may include a 3'UTR, 5'UTR, exon, intron, exon/intron junction, coding region, translation initiation region, translation termination region, or other defined nucleic acid region. Regions structurally defined for DMPK can be obtained by accession numbers from sequence databases such as NCBI, and such information is incorporated herein by reference. In certain embodiments, the target region may comprise a sequence from the 5'target site of one target segment within the target region to the 3'target site of another target segment within the target region.

Targeting involves the determination of at least one target segment where the antisense compound hybridizes and the desired effect occurs. In certain embodiments, the desired effect is a decrease in the level of the mRNA target nucleic acid. In certain embodiments, the desired effect is a decrease in the level of the protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

The target region may contain one or more target segments. Multiple target segments within the target region may overlap. Alternatively, multiple target segments may not overlap. In certain embodiments, the target segment within the target region is separated by no more than about 300 nucleotides. In certain embodiments, the target segment within the target region is 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides on the target nucleic acid, or any two of the above values. It is separated by a number of nucleotides, which is a range defined by values. In certain embodiments, the target segment within the target region is separated by no more than 5 or no more than about 5 nucleotides on the target nucleic acid. In certain embodiments, the target segment is contiguous. A target region is contemplated that is defined by a range with a starting nucleic acid that is any of the 5'target sites or 3'target sites listed herein.

Suitable target segments can be found within the 5'UTR, coding region, 3'UTR, intron, exon or exon/intron junction. Target segments containing a start or stop codon are also suitable target segments. Suitable target segments may specifically exclude certain structurally defined regions, such as start codons or stop codons.

Determination of suitable target segments may include comparison of the sequence of the target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify similar regions among various nucleic acids. Such comparison may prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than the selected target nucleic acid (ie, non-target or off-target sequences).

There may be a change in the activity of the antisense compound within the active target region (eg, defined by the percent reduction in the target nucleic acid level). In certain embodiments, a decrease in DMPK mRNA level indicates inhibition of DMPK protein expression. A decrease in the DMPK protein level also indicates inhibition of target mRNA expression. In addition, phenotypic changes, such as reduction in dystonia or reduction in splice opacity, can result from inhibition of DMPK mRNA and/or protein expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a DMPK nucleic acid. The most common mechanisms of hybridization involve hydrogen bonding between complementary nucleobases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding). do.

Hybridization can occur under a variety of conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecule to be hybridized.

Methods for determining whether a sequence specifically hybridizes to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3 ^rd Ed., 2001). In certain embodiments, antisense compounds provided herein are specifically hybridizable with DMPK nucleic acids.

Complementarity

Antisense compounds and target nucleic acids are capable of hydrogen reaction of a sufficient number of nucleobases of the antisense compound with the corresponding nucleobases of the target nucleic acid to produce the desired result (e.g., antisense inhibition of the target nucleic acid, e.g., DMPK nucleic acid). If present, they are complementary to each other.

Antisense compounds can hybridize on one or more segments of a DMPK nucleic acid, such that intervening or adjacent segments are not associated with hybridization events (e.g., loop structures, mismatches, or hairpin structures).

In certain embodiments, an antisense compound provided herein, or a specified portion thereof, is at least 70%, 80%, 85%, 86%, 87%, 88%, to a DMPK nucleic acid, a target region, a target segment, or a specified portion thereof, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary. In certain embodiments, the antisense compound is at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% to a DMPK nucleic acid, a target region, a target segment, or a specified portion thereof. , At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary, as described herein At least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 of the nucleobase sequence of any of the exemplary antisense compounds. It contains contiguous nucleobases (eg, at least 8 contiguous nucleobases of the nucleobase sequence listed in any one of SEQ ID NOs: 12-156, 160-770 and 774-792). The percent complementarity of an antisense compound with a target nucleic acid can be determined using conventional methods, which are measured throughout the antisense compound.

For example, 18 of the 20 nucleobases of an antisense compound are complementary to the target region, and thus an antisense compound that hybridizes specifically exhibits 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered or scattered with respect to the complementary nucleobases, and need not be contiguous to each other or to complementary nucleobases. As described above, an antisense compound having a length of 18 nucleobases having 4 (4) non-complementary nucleobases on the flanks of 2 regions of complete complementarity with the target nucleic acid has 77.8% complementarity with the target nucleic acid as a whole, and thus the present It belongs to the scope of the invention. The percent complementarity of the region of the antisense compound and the target nucleic acid can be determined conventionally using the BLAST program (basic local alignment search tool) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990 , 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity is determined by the Gap program (Wisconsin Sequence) using default settings using, for example, Smith and Waterman's algorithm (Adv. Appl. Math., 1981, 2, 482 489). Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.).

In certain embodiments, an antisense compound provided herein or a specified portion thereof is completely complementary (ie, 100% complementary) to the target nucleic acid or specified portion thereof. For example, an antisense compound can be completely complementary to a DMPK nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means that each nucleobase of an antisense compound is capable of forming an exact base pair with the corresponding nucleobase of the target nucleic acid. For example, a 20 nucleobase antisense compound is completely complementary to a target sequence that is 400 nucleobases long as long as there are corresponding 20 nucleobase portions of the target nucleic acid that are completely complementary to the antisense compound. Full complementarity can also be used with respect to a specified portion of the first and/or second nucleic acid. For example, a portion of the 20 nucleobases of a 30 nucleobase antisense compound may be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase moieties of the 30 nucleobase oligonucleotides are completely complementary to the target sequence if each nucleobase has a corresponding 20 nucleobase moiety that is complementary to the 20 nucleobase moiety of the antisense compound. It's the enemy. At the same time, a total of 30 nucleobase antisense compounds can be completely complementary to the target sequence depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The position of the non-complementary nucleobase may be at the 5'end or 3'end of the antisense compound. Alternatively, non-complementary nucleobases or nucleobases may be present at the internal position of the antisense compound. When two or more non-complementary nucleobases are present, they can be contiguous (i.e. linked) or non-contiguous. In one embodiment, the non-complementary nucleobase is located in the wing segment of the gapmer antisense oligonucleotide.

In certain embodiments, an antisense compound having a length of 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleobases or less nucleobases is a target nucleic acid, e.g., a DMPK nucleic acid, Or 4 or less, 3 or less, 2 or less, or 1 or less non-complementary nucleobase(s) relative to the specified portion thereof.

In certain embodiments, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases are in length, or Antisense compounds with a nucleobase length of less than or equal to a target nucleic acid, e.g., a DMPK nucleic acid, or a specified portion thereof, is 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. Contains non-complementary nucleobase(s) of.

Antisense compounds provided herein also include those that are complementary to a portion of the target nucleic acid. As used herein, the term “portion” refers to a defined number of contiguous (ie linked) nucleobases within a region or segment of a target nucleic acid. "Part" also means a defined number of consecutive nucleobases of an antisense compound. In certain embodiments, the antisense compound is complementary to at least 8 nucleobase moieties of the target segment. In certain embodiments, the antisense compound is complementary to at least 10 nucleobase moieties of the target segment. In certain embodiments, the antisense compound is complementary to at least 15 nucleobase moieties of the target segment. At least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more nucleobase portions of the target segment, Or antisense compounds that are complementary to the range defined by any two of the above values are also contemplated.

sameness

Antisense compounds provided herein may also have a defined percent identity to a compound represented by a particular nucleotide sequence, a sequence number, or a particular Isis number, or a portion thereof. Antisense compounds as used herein are identical to the sequences disclosed herein if such antisense compounds have the same nucleobase pairing ability. For example, RNA containing uracil instead of thymidine within the disclosed DNA sequence is considered identical to the DNA sequence as uracil and thymidine pair with adenine. Shortened and extended forms of the antisense compounds described herein as well as compounds having bases that are not identical compared to the antisense compounds provided herein are also contemplated. Bases that are not identical may exist adjacent to each other or may be dispersed throughout the antisense compound. The percent identity of an antisense compound is calculated according to the number of bases having the same base pairing compared to the sequence being compared.

In certain embodiments, the antisense compound or portion thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least one or more of the exemplary antisense compounds or SEQ ID NOs disclosed herein, or portions thereof. 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

transform

Nucleosides are base-sugar combinations. The nucleobase (also known as base) portion of the nucleoside is usually a heterocyclic base moiety. A nucleotide is a nucleoside that further contains a phosphate group covalently bonded to the sugar moiety of the nucleoside. For nucleosides comprising pentofuranosyl sugars, the phosphate group may be attached to the 2', 3'or 5'hydroxyl moiety of the sugar. Oligonucleotides are formed through covalent bonds of nucleosides adjacent to each other to form linear polymeric oligonucleotides. Within the oligonucleotide structure, the phosphate group is usually referred to as forming an internucleoside linkage of the oligonucleotide.

Modifications to antisense compounds include internucleoside linkages, substitutions or changes to sugar moieties or nucleobases. Modified antisense compounds are often preferred over the natural form due to desired properties, such as improved cellular uptake, improved affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be used to increase the binding affinity of shortened or truncated antisense oligonucleotides to target nucleic acids. As a result, equivalent results can often be obtained using shorter antisense compounds with such chemically modified nucleosides.

Modified nucleoside linkage

The linkage between the naturally occurring nucleosides of RNA and DNA is a phosphodiester linkage from 3′ to 5′. One or more modified, i.e., non-naturally occurring internucleoside binding, due to the desired properties, e.g., improved cellular uptake, improved affinity for the target nucleic acid, and increased stability in the presence of nucleases. Antisense compounds having are often more screened than antisense compounds having naturally occurring internucleoside linkages.

Oligonucleotides having a modified internucleoside bond include an internucleoside bond having a phosphorus atom as well as an internucleoside bond not having a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, phospholiesters, methylphosphonates, phosphoramidates and phosphorothioates. Phosphorus-containing bonds and methods of making phosphorus-free bonds are well known.

In certain embodiments, antisense compounds targeting DMPK nucleic acids comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkage is a phosphorothioate linkage. In certain embodiments, each internucleoside linkage of the antisense compound is a phosphorothioate internucleoside linkage.

Modified sugar moiety

Antisense compounds of the present invention may optionally contain one or more nucleosides with modified sugar groups. Such sugar modified nucleosides can provide improved nuclease stability, increased binding affinity, or some other advantageous biological property for antisense compounds. In certain embodiments, the nucleoside comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituted groups (including 5'and 2'substituents), non-double (non-double) to form bicyclic nucleic acids (BNA). geminal) bridging of ring atoms, S, N(R) or C(R ₁ )(R ₂ )(R, R ₁ and R ₂ of the ribosyl ring oxygen atom are each independently H, C _1- C ₁₂ alkyl or a protecting group), and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (PCT international application published on August 21, 2008 for other disclosed 5',2'-bis substituted nucleosides). WO 2008/101157) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see US Patent Application No. US2005-0130923 published June 16, 2005) or alternatively 5'-substitution of BNA (see PCT International Application WO 2007/134181 published on November 22, 2007, wherein LNA is substituted with, for example, 5'-methyl or 5'-vinyl group). .

Examples of nucleosides with modified sugar moieties include, but are not limited to, 5'-vinyl, 5'-methyl ( R or S ), 4'-S, 2'-F, 2'-OCH ₃ , 2'-OCH ₂ CH ₃ , 2'-OCH ₂ CH ₂ F and 2'-O(CH ₂ ) ₂ OCH ₃ substituents. Substitution at the 2'position is also allyl, amino, azido, thio, O-allyl, OC ₁ -C ₁₀ alkyl, OCF ₃ , OCH ₂ F, O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(R _m )(R _n ), O-CH ₂ -C(=O)-N(R _m )(R _n ) and O-CH ₂ -C(=O)-N(R ₁ )-( CH ₂ ) ₂ -N(R _m )(R _n ), wherein each R ₁ , R _m and R _n is independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

Examples of bicyclic nucleic acids (BNA) include, but are not limited to, nucleosides comprising a bridge between 4'and 2'ribosyl ring atoms. In certain embodiments, antisense compounds provided herein have a bridge of 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'-CH(CH ₂ OCH ₃ )-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845 issued July 15, 2008) ; 4′-C(CH ₃ )(CH ₃ )-O-2′ (and analogs thereof, see PCT/US2008/068922 published on Jan. 8, 2009 as WO/2009/006478); 4'-CH ₂ -N(OCH ₃ )-2' (and analogs thereof, see PCT/US2008/064591 published on Dec. 11, 2008 as WO/2008/150729); 4'-CH ₂ -ON(CH ₃ )-2' (see published US patent application US2004-0171570 published Sep. 2, 2004); _{4′-CH 2} -N(R)-O-2′ in which R is H, C ₁ -C ₁₂ alkyl or a protecting group (see US Pat. No. 7,427,672 issued Sep. 23, 2008); 4'-CH ₂ -C(H)(CH ₃ )-2' (see Chattopadhyaya et al., J. Org. Chem. , 2009, 74 , 118-134); And 4'-CH ₂ -C(=CH ₂ )-2' (and analogs thereof, see PCT/US2008/066154 published as WO 2008/154401 on December 8, 2008). It contains one or more BNA nucleosides.

Additional bicyclic nucleosides have been reported in published literature (e.g., Srivastava et al., J. Am. Chem. Soc , 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids. Research , 2003, 21 , 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs , 2001, 2 , 558-561; Braasch et al., Chem. Biol , 2001, 8 , 1-7; Orum et al. ., Curr. Opinion Mol. Ther. , 2001, 3 , 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci. USA , 2000, 97 , 5633-5638; Singh et al., Chem. Commun . , 1998, 4 , 455-456; Koshkin et al., Tetrahedron , 1998, 54 , 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett. , 1998, 8 , 2219-2222; Singh et al. , J. Org. Chem. , 1998, 63 , 10035-10039; U.S. Patent Nos. 7,399,845; 7,053,207; 7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication Nos: US2008-0039618; US2007-0287831; US2004-0171570; U.S. Patent Application Nos. 12/129,154; 61/099,844; 61/097,787; 61/086,231; 61/ 056,564; 61/026,998; 61/026,995; 60/989,574; international application WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226; and PCT International Filing Number: PCT/US2008/068922; PCT/US2008/066154; And PCT/US2008/064591). Each of the bicyclic nucleosides may be prepared to have one or more stereochemical sugar forms including, for example, α-L-ribofuranose and β-D-ribofuranose (as WO 99/14226). See PCT International Application PCT/DK98/00393 published March 25, 1999).

In certain embodiments, the bicyclic nucleoside is -[C(R _a )(R _b )] _n -, -C(R _a )=C(R _b )-, -C(R _a )=N- , -C(=NR _a )-, -C(=O)-, -C(=S)-, -O-, -Si(R _a ) ₂ -, -S(=O) _x -and -N (R _a )-includes a bridge between the 4'and 2'carbon atoms of the pentofuranosyl sugar moiety, including, but not limited to, a bridge comprising 1 or 1 to 4 bonded groups independently selected from -, wherein Where x is 0, 1 or 2 and n is 1, 2, 3 or 4; Each R _a and R _b is independently H, protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl , C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted Heteroaryl, C ₅ -C ₇ alicyclic radical, substituted C ₅ -C ₇ alicyclic radical, halogen, OJ ₁ , NJ ₁ J ₂ , SJ ₁ , N ₃ , COOJ ₁ , acyl (C(=O )-H), substituted acyl, CN, sulfonyl (S(=O) ₂ -J ₁ ) or sulfoxyl (S(=O)-J ₁ ), and each of J ₁ and J ₂ is independently H , C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ Alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, acyl (C(=O)-H), substituted acyl, heterocycle radical, substituted heterocycle radical, C ₁ -C ₁₂ amino Alkyl, substituted C ₁ -C ₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridging of the bicyclic sugar moiety is -[C(R _a )(R _b )] _n -, -[C(R _a )(R _b )] _n -O-, -C(R _a R _b )-N(R)-O- or -C(R _a R _b )-ON(R)-. In certain embodiments, the bridge is 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -O-2' , 4'-(CH ₂ ) ₂ -O-2', 4'-CH ₂ -ON(R)-2' and 4'-CH ₂ -N(R)-O-2-, and in the above formula, each R is independently H, a protecting group or C ₁ -C ₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric forms. For example, a nucleoside including a 4'-(CH ₂ )-O-2' bridge may exist in an α-L form or a β-D form. Previously, α-L-methyleneoxy (4'-CH ₂ -O-2') BNA was incorporated into antisense oligonucleotides showing antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21 , 6365-6372 ).

In certain embodiments, bicyclic nucleosides include those having a 4'to 2'bridge, wherein the bridge is, by way of non-limiting example, α-L-4'-(CH ₂ )-O- 2', β-D-4'-CH ₂ -O-2', 4'-(CH ₂ ) ₂ -O-2', 4'-CH ₂ -ON(R)-2', 4'-CH ₂ -N(R)-O-2', 4'-CH(CH ₃ )-O-2', 4'-CH ₂ -S-2', 4'-CH ₂ -N(R)-2' , 4'-CH ₂ -CH(CH ₃ )-2' and 4'-(CH ₂ ) ₃ -2', wherein R is H, a protecting group or C ₁ -C ₁₂ alkyl.

In certain embodiments, bicyclic nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

-Q _a -Q _b -Q _c -is -CH ₂ -N(R _c )-CH ₂ -, -C(=O)-N(R _c )-CH ₂ -, -CH ₂ -ON(R _c )-, -CH ₂ -N(R _c )-O- or -N(R _c )-O-CH ₂ ;

R _c is a C ₁ -C ₁₂ alkyl or amino protecting group;

T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to the support medium.

In certain embodiments, bicyclic nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to the support medium;

Z _a is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C _2- C ₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups is independently halogen, oxo, hydroxyl, OJ _c , NJ _c J _d , SJ _c , N ₃ , OC(=X)J _c and NJ _e C(=X)NJ _c Is mono or poly substituted with a substituent selected from _{J d , wherein each J c} , J _d and J _e is independently H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl, and X is O or NJ _c .

In certain embodiments, bicyclic nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to the support medium;

Z _b is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C _2- C ₆ alkynyl or substituted acyl (C(=O)-).

In certain embodiments, bicyclic nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to the support medium;

R _d is C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C _2- C ₆ alkynyl;

Each of q _a , q _b , q _c and q _d is independently H, halogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxyl, substituted C ₁ -C ₆ alkoxyl, acyl, substituted acyl, C ₁ -C ₆ aminoalkyl or substituted C ₁ -C ₆ aminoalkyl.

In certain embodiments, bicyclic nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to the support medium;

q _a , q _b , q _e and q _f are each independently hydrogen, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ Alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxy, substituted C ₁ -C ₁₂ alkoxy, OJ _j , SJ _j , SOJ _j , SO ₂ J _j , NJ _j J _k , N ₃ , CN, C(=O)OJ _j , C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H )C(=NH)NJ _j J _k , N(H)C(=O)NJ _j J _k or N(H)C(=S)NJ _j J _k ;

q _e and q _f together are =C(q _g )(q _h );

q _g and q _h are each independently H, halogen, C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl.

Synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides _{with 4'-CH 2} -O-2' bridges with oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron , 1998, 54 , 3607-3630). The synthesis of bicyclic nucleosides is also described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides with 4'to 2'bridging groups such as 4'-CH ₂ -O-2' and 4'-CH ₂ -S-2' have also been prepared (Kumar et al. al., Bioorg. Med. Chem. Lett , 1998, 8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Moreover, the synthesis of 2'-amino-BNA, a novel conformationally limited high-affinity oligonucleotide analogue, has also been described in the art (Singh et al., J Org. Chem. , 1998, 63 , 10035-10039. ). In addition, 2'-amino- and 2'-methylamino-BNA were prepared, and the thermal stability of the duplex with complementary RNA and DNA strands was previously reported.

In certain embodiments, bicyclic nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to the support medium;

Each of q _i , q _j , q _k and q _l is independently H, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxyl, substituted C ₁ -C ₁₂ alkoxyl, OJ _j , SJ _j , SOJ _j , SO ₂ J _j , NJ _j J _k , N ₃ , CN, C(=O)OJ _j , C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH)NJ _j J _k , N(H)C(=O)NJ _j J _k or N(H)C(=S)NJ _j J _k ;

q _i and q _j or q _l and q _k together are =C(q _g )(q _h ), where q _g and q _h are each independently H, halogen, C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl.

One carbocyclic bicyclic nucleoside with a 4'-(CH ₂ ) ₃ -2' bridge and an alkenyl analog bridge 4'-CH=CH-CH ₂ -2' has been described (Frier et al. , Nucleic Acids Research , 1997, 25(22) , 4429-4443 and Albaek et al., J. Org. Chem. , 2006, 71 , 7731-7740). Synthesis and preparation of carbocyclic bicyclic nucleosides along with oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc . 2007, 129(26) , 8362-8379). .

In certain embodiments, the bicyclic nucleoside is (A) α-L-methyleneoxy (4′-CH ₂ -O-2′) BNA, (B) β-D-methyleneoxy ( 4'-CH ₂ -O-2') BNA, (C) ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') BNA, (D) aminooxy (4'-CH ₂ -ON(R )-2') BNA, (E) oxyamino (4'-CH ₂ -N(R)-O-2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH ₃ )-O -2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH ₂ -S-2') BNA, (H) methylene-amino (4'-CH ₂ -N(R)-2') BNA, (I) methyl carbocyclic (4'-CH ₂ -CH(CH ₃ )-2') BNA, (J) propylene carbocyclic (4'-(CH ₂ ) ₃ -2') BNA and (K) vinyl BNA.

Wherein Bx is a base moiety and R is independently H, a protecting group, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy.

In certain embodiments, nucleosides are modified by substitution of a ribosyl ring with a sugar surrogate. Such modifications can be made with a surrogate ring system of the ribosyl ring (sometimes referred to as a DNA analog), e.g., a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring, e.g. the formula Including, but not limited to, having one of:

In certain embodiments, sugar substitutes are selected having the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T ₃ and T ₄ are each independently an internucleoside linking group that connects a tetrahydropyran nucleoside analog to an _{oligomeric compound, or one of T 3} and T ₄ is an oligomeric compound or a tetrahydropyran nucleoside analog to an oligonucleotide. Is an internucleoside linking group connecting T ₃ and T ₄ , and the rest of T 3 and T 4 are H, a hydroxyl protecting group, a bonded conjugate group, or a 5′ or 3′-terminal group;

q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are each independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, Substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl;

One of R ₁ and R ₂ is hydrogen, and the rest is halogen, substituted or unsubstituted alkoxy, NJ ₁ J ₂ , SJ ₁ , N ₃ , OC(=X)J ₁ , OC(=X)NJ ₁ J ₂ , NJ ₃ C(=X)NJ ₁ J ₂ and CN, wherein X is O, S or NJ ₁ , and each J ₁ , J ₂ and J ₃ is independently H or C ₁ -C ₆ alkyl to be.

In certain embodiments, q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are each H. In certain embodiments, at least one of _{q 1} , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q _{7 is not H.} In certain embodiments, at least one of _{q 1} , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q _{7 is methyl.} In certain embodiments, THP nucleosides are provided, wherein one of _{R 1} and R _{2 is F.} In certain embodiments, R ₁ is fluoro and R ₂ is H; R ₁ is methoxy, R ₂ is H, R ₁ is methoxyethoxy, and R ₂ is H.

Such sugar substitutes include, but are not limited to, those referred to in the art as hexitol nucleic acids (HNA), altitol nucleic acids (ANA) and mannitol nucleic acids (MNA) (Leumann, CJ, Bioorg. & Med. Chem. . , 2002, 10 , 841-854).

In certain embodiments, the antisense compound comprises one or more modified cyclohexenyl nucleosides that are nucleosides having a 6-membered cyclohexenyl instead of a pentofuranosyl moiety of the naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (e.g., commonly owned PCT application WO 2010/036696 published on April 10, 2010, Robeyns et al., J. Am. Chem. Soc , 2008, 130(6) , 1979-1984; Horvath et al., Tetrahedron Letters , 2007, 48 , 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc , 2007, 129(30) , 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids , 2005, 24(5-7) , 993-998; Nauwelaerts et al., Nucleic Acids Research , 2005, 33( 8) , 2452-2463; Robeyns et al., Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111- 2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem ., 2003, 68 , 4499-4505; Verbeure et al., Nucleic Acids Research, 2001 , 29(24), 4941-4947; Wang et al., J. Org. Chem. , 2001, 66 , 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids , 2001, 20(4-7) , 785-788; Wang et al., J. Am. Chem. , 2000, 122, 8595-8602; published PCT application, WO 06/047842; and See published PCT application WO 01/049687; Each of these documents is incorporated herein by reference in its entirety). Certain modified cyclohexenyl nucleosides have the formula:

In the above formula,

Bx is a heterocyclic base moiety;

T ₃ and T ₄ are each independently an internucleoside linking group linking a cyclohexenyl nucleoside analog to an _{antisense compound, or one of T 3} and T ₄ is a tetrahydropyran nucleoside analog linking an antisense compound. Is an internucleoside linking group, and the _{rest of T 3 and T 4} are H, a hydroxyl protecting group, a bonded conjugate group, or a 5'or 3'-terminal group; q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , q ₇ , q ₈ and q ₉ are each independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ alkynyl or other sugar substituent.

Many other bicyclic and tricyclic sugar surrogate ring systems that can be used to modify nucleosides for incorporation into antisense compounds are also known in the art (see, for example, Leumann, Christian J. , Bioorg. & Med. Chem. , 2002, 10 , 841-854). These ring systems can undergo various additional substitutions to enhance activity.

Methods of making modified sugars are well known to those skilled in the art. Some representative US patents teaching the preparation of such modified sugars are described in US Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,387,878; 5,393,878; 5,446,137; 5,446,137; 5,466,786; 5,514,785; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,591,722; 5,597,909; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032, and international applications PCT/US2005/019219, filed on June 2, 2005 and published as WO 2005/121371 on December 22, 2005, but are not limited thereto. , Each of the above patents is incorporated herein by reference in its entirety.

In nucleotides with modified sugar moieties, nucleobase moieties (natural, modified or combinations thereof) are maintained for hybridization with appropriate nucleic acid targets.

In certain embodiments, antisense compounds targeting DMPK nucleic acids comprise one or more nucleotides with modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleotides are arranged within a gapmer motif.

Modified nucleobase

Nucleobase (or base) modifications or substitutions are structurally distinguishable from naturally occurring or synthetic unmodified nucleobases, but functionally interchangeable. Both natural and modified nucleobases can participate in hydrogen bonding. Such nucleobase modifications can provide antisense compounds with nuclease stability, binding affinity, or some other advantageous biological property. Modified nucleobases include synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability to 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional unmodified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenin, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiotimine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ₃ ) uracil and cytosine and pyrimidine base Other alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyl Adenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. .

Heterocyclic base moieties also include those in which purine or pyrimidine bases have been replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. can do. Particularly useful nucleobases for increasing the binding affinity of antisense compounds are 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, such as 2 aminopropyladenine. , 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeting DMPK nucleic acids comprise one or more modified nucleobases. In certain embodiments, gap-expanded antisense oligonucleotides targeting DMPK nucleic acids comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

Compositions and methods for formulating pharmaceutical compositions

Antisense oligonucleotides can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The composition and method for formulating the pharmaceutical composition depends on a number of criteria including, but not limited to, the route of administration, the extent of the disease, or the dose administered.

Antisense compounds targeting DMPK nucleic acids can be used in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in compositions delivered parenterally. Thus, in one embodiment, a pharmaceutical composition comprising an antisense compound targeting a DMPK nucleic acid and a pharmaceutically acceptable diluent is used in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the intisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds are any pharmaceutically acceptable salts, esters, or any pharmaceutically acceptable salts, esters or the above capable of providing (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans. Salts of esters, or any other oligonucleotides. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of antisense compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

Prodrugs may include the incorporation of additional nucleosides at one or both ends of an antisense compound that is cleaved by an endogenous nuclease in the body to form an active antisense compound.

Conjugated antisense compounds

Antisense compounds may be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups generally attached to one or both ends of the antisense compound to enhance properties such as, for example, nuclease stability. The cap structure is included in the stabilizing group. These terminal modifications protect antisense compounds with terminal nucleic acids from exonuclease degradation, which can also aid in delivery and/or localization in cells. The cap may be present at the 5'-end (5'-cap) or the 3'-end (3'-cap), or may be present at both ends. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Additionally, 3'and 5'-stabilizing groups that can be used to cap one or both ends of an antisense compound to provide nuclease stability are disclosed in WO 03/004602, published on January 16, 2003. Includes.

Cell culture and antisense compound treatment

The effect of antisense compounds on the level, activity or expression of DMPK nucleic acids can be tested in vitro in a variety of cell types. Cell types used in the assay were commercially available (e.g., American Type Culture Collection, Manassas, Virginia; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walker, Maryland). Sville City), and cells are cultured according to the manufacturer's instructions using commercially available reagents (eg, Invitrogen Life Technologies, Carlsbad, CA). Exemplary cell types include, but are not limited to, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells.

In vitro testing of antisense oligonucleotides

Described herein are methods for the treatment of cells with antisense oligonucleotides, which can be suitably modified for treatment with other antisense compounds.

In general, cells are treated with antisense oligonucleotides when cells reach about 60-80% confluence during culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, Calif.). The antisense oligonucleotide is LIPOFECTIN (registered trademark) in OPTI-MEM (registered trademark) 1 to achieve the desired final concentration and LIPOFECTIN (registered trademark) concentration of the antisense oligonucleotide, typically in the range of 2 to 12 ug/mL per 100 nM antisense oligonucleotide. Trademark) (Invitrogen, Carlsbad, CA).

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTAMINE 2000 (registered trademark) (Invitrogen, Carlsbad, Calif.) in OPTI-MEM (registered trademark) 1 reduced serum medium, and typically in the range of 2 to 12 ug/mL per 100 nM antisense oligonucleotide. The desired concentration and LIPOFECTAMINE® concentration of the antisense oligonucleotide of are achieved.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with Cytofectin (registered trademark) (Invitrogen, Carlsbad, Calif.) in OPTI-MEM (registered trademark) 1 reduced serum medium, and typically in the range of 2 to 12 ug/mL per 100 nM antisense oligonucleotide. The desired concentration of antisense oligonucleotide and Cytofectin® concentration are achieved.

Another technique used to introduce antisense oligonucleotides into cultured cells involves electroporation.

Cells are treated with antisense oligonucleotides by conventional methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, where the RNA or protein level of the target nucleic acid is determined by methods known in the art and described herein. In general, when processing is performed with multiple iterations, the data is presented as the average of the iterations.

The concentration of the antisense oligonucleotide used varied from cell line to cell line. Methods for determining the optimal antisense oligonucleotide for a particular cell line are well known in the art. Antisense oligonucleotides are typically used in a concentration ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000 (registered trademark), Lipofectin or Cytofectin. Antisense oligonucleotides are used in high concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, eg, using the TRIZOL® reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocol.

Analysis of inhibition of target level or expression

Inhibition of the level or expression of DMPK nucleic acids can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR) or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also common in the art. Quantitative real-time PCR is a commercially available ABI PRISM® 7600, 7700 or 7900 sequence detection system available from PE-Applied Biosystems (Foster City, CA) and used according to the manufacturer's instructions. It can be conveniently achieved by using.

Quantitative real-time PCR analysis of target RNA level

Quantification of target RNA levels can be achieved by quantitative real-time PCR using an ABI PRISM® 7600, 7700 or 7900 sequence detection system (PE-Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. . Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction to generate complementary DNA (cDNA) that is used later as a substrate for real-time PCR amplification. RT and real-time PCR reactions are performed continuously in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT, real-time PCR reaction is performed by a method well known to those skilled in the art.

The target amount of a gene (or RNA) obtained by real-time PCR is determined by using the expression level of a gene with constant expression, for example, cyclophilin A, or RIBOGREEN (registered trademark) (Invitrogen, Inc. Carlsbad, CA). City) is standardized by quantifying total RNA. Cyclophilin A expression is quantified by real-time PCR, which is performed simultaneously with the target, or in multiples, or independently. Total RNA is quantified using RIBOGREEN (registered trademark) RNA quantification reagent (Invitrogen, Inc., Eugene, Oregon). The method of RNA quantification by RIBOGREEN (registered trademark) is taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). The CYTOFLUOR® 4000 machine (PE Applied Biosystems) is used to measure RIBOGREEN® fluorescence.

Probes and primers are designed to hybridize to DMPK nucleic acids. Methods for designing real-time PCR probes and primers are well known in the art and may include the use of software such as PRIMER EXPRESS® software (Applied Biosystems, Foster City, CA).

Protein level analysis

Antisense inhibition of DMPK nucleic acids can be assessed by measuring DMPK protein levels. The protein level of DMPK is determined in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay. (E.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence activated cell sorting (FACS). Antibodies specific for the target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham City, Michigan), or these are conventional monoclonal or polyclonal antibodies well known in the art. It can be manufactured through a production method.

In vivo testing of antisense compounds

Antisense compounds, such as antisense oligonucleotides, are tested in animals to assess their ability to inhibit the expression of DMPK and produce phenotypic changes. The test can be performed in a normal animal, or in an experimental disease model, for example, an HSA ^LR mouse model of muscular dystrophy (DM1).

The HSA ^LR mouse model is an established model for DM1 (Mankodi, A. et al. Science. 289: 1769, 2000). Mice have a human skeletal actin ( hACTA1 ) transgene with 220 CTG repeats inserted into the 3'UTR of the gene. The hACTA1- CUG ^exp transcript accumulates in the nuclear foci in skeletal muscle, which causes dystonia similar to that of human DM1 (Mankodi, A. et al. Mol. Cell 10: 35, 2002; Lin. , X. et al. Hum. Mol. Genet. 15: 2087, 2006). Therefore, ^{improvement of DM1 symptoms in HSA LR} mice by antisense inhibition of hACTA1 transgene is expected to predict the improvement of similar symptoms in human patients by antisense inhibition of DMPK transcripts.

^{Expression of CUG exp} RNA in mice results in extensive remodeling of muscle transcripts, many of which are regenerated by removal of MBNL1. Therefore, ^{normalization of transcripts in HSA LR} mice is expected to predict normalization of human transcripts in DM1 patients by antisense inhibition of DMPK transcripts.

For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration. After a period of treatment with antisense oligonucleotides, RNA is isolated from the tissue and changes in DMPK nucleic acid expression are measured. Changes in the DMPK protein level are also measured.

Splicing

Myosonic dystrophy (DM1) is caused by expansion of CTG repeats within the 3'untranslated region of the DMPK gene (Brook, JD et al. Cell. 68: 799, 1992). These mutations cause RNA dominance, a process in which the expression of RNA containing extended CUG repeats (CUGexp) induces cell dysfunction (Osborne RJ and Thornton CA., Human Molecular Genetics. , 2006 , 15(2)) : R162-R169). These CUGexps are maintained as the nuclear foci of skeletal muscle (Davis, BM et al. Proc. Natl. Acad. Sci. USA 94:7388, 1997). Accumulation of CUGexp in the nuclear focal point results in sequestration of poly(CUG)-binding proteins, such as Muscleblind-like 1 (MBLN1) (Miller, JW et al. EMBO J. 19: 4439, 2000). MBLN1 is a splicing factor, which regulates the splicing of genes such as Serca1, CIC-1, Titin and Zasp. Thus, sequestration of MBLN1 by CUGexp triggers a misregulated alternative splicing of exons of genes normally regulated by MBLN1 (Lin, X. et al. Hum. Mol. Genet. 15: 2087, 2006). Correction of alternative splicing in animals exhibiting this dysregulation, such as in DM1 patients and HSALR mouse models, is a useful indicator of the efficacy of treatments including treatment with antisense oligonucleotides.

Specific biomarker

DM1 severity in the mouse model is determined at least in part by the level ^{of CUG exp transcript accumulation in the nucleus or nuclear foci.} A useful physiological marker for DM1 severity is the development of high-frequency performance of involuntary action potentials (myotonia).

Specific indicator

In certain embodiments, provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the subject has type 1 muscular dystrophy (DM1).

Accordingly, provided herein is a method of ameliorating the symptoms associated with type 1 muscular dystrophy in a subject in need of ameliorating the symptoms associated with type 1 muscular dystrophy. In certain embodiments, a method of reducing the rate of onset of symptoms associated with type 1 muscular dystrophy is provided. In certain embodiments, a method of reducing the severity of symptoms associated with type 1 muscular dystrophy is provided. In certain embodiments, symptoms associated with DM1 include muscle stiffness, dystonia, distal distal weakness, facial and jaw muscles weakness, difficulty swallowing, loss of eyelid strength (ptosis), neck muscles. Weakness, weakness of the arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory failure, irregular heartbeat, heart muscle damage, numbness, insulin resistance, and cataracts. In children, symptoms can also be developmental delays, learning disabilities, language and speech problems, and personality development problems.

In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound targeting the DMPK nucleic acid.

In certain embodiments, administration of an antisense compound targeting a DMPK nucleic acid is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%. , At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% Or at least about 99%, or to a range defined by any two of the above values.

In certain embodiments, a pharmaceutical composition comprising an antisense compound targeting DMPK is used for the manufacture of a medicament for treating a patient suffering from or susceptible to type 1 myotonic dystrophy.

In certain embodiments, the methods described herein administer a compound comprising a modified oligonucleotide having a contiguous nucleobase moiety as described herein of the sequences listed in SEQ ID NOs: 12-156, 160-770 and 774-792. Includes that.

administration

In certain embodiments, compounds and compositions as described herein are administered parenterally.

In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent infusion. In certain embodiments, the infused pharmaceutical agent is delivered using a pump. In certain embodiments, parenteral administration is by injection (eg, bolus injection). Injections can be delivered using a syringe.

Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration or intracranial administration, such as intrathecal or intraventricular administration. Administration may be continuous, or chronic, or short, or intermittent.

In certain embodiments, the administration is subcutaneous, intravenous, intracranial, intraventricular, intrathecal or another administration that results in a systemic effect of an oligonucleotide (systemic administration is characterized by a systemic effect, i. ) Or delivery to the CNS or CSF.

The duration of action as measured by inhibition of alpha 1 actin and reduction of dystonia in the HSA ^LR mouse model of DM1 extends in muscle tissue including the quadriceps muscle, calf muscle and tibialis anterior muscle (see Examples below). do. Subcutaneous injection of antisense oligonucleotides for 4 weeks results in inhibition of alpha 1 actin by at least 70% in the quadriceps, calf and anterior tibialis muscles of ^{HSA LR} mice for at least 11 weeks (77 days) after the end of dosing. Subcutaneous injection of antisense oligonucleotides for 4 weeks results in the removal of myotonia in the quadriceps, calf and anterior tibialis in ^{HSA LR} mice for at least 11 weeks (77 days) after the end of administration.

In certain embodiments, delivery of a compound or composition as described herein results in at least 70% downregulation of the target mRNA and/or target protein for at least 77 days. In certain embodiments, the delivery of a compound or composition as described herein is at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days. Days, at least 75 days, at least 76 days, at least 77 days, at least 78 days, at least 79 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or of the target mRNA and/or target protein for at least 115 days, at least 120 days, at least 1 year 100% downregulation occurs.

In certain embodiments, the antisense oligonucleotide is delivered by injection or infusion once daily for 77 days. In certain embodiments, the antisense oligonucleotide is delivered by injection or infusion once a month, once every two months, once every three months, once every six months, twice a year, or once a year.

Specific combination therapy

In certain embodiments, a first agent comprising a modified oligonucleotide of the invention is co-administered with one or more second agents. In certain embodiments, this second agent is designed to treat the same type 1 myotonic dystrophy as the first agent described herein. In certain embodiments, this second agent is designed to treat a different disease, disorder or condition than the first agent described herein. In certain embodiments, this second agent is designed to treat undesired side effects of one or more pharmaceutical compositions as described herein. In certain embodiments, the second agent is co-administered with the first agent to treat the undesired consequences of the first agent. In certain embodiments, the second agent is co-administered with the first agent to produce a combination effect. In certain embodiments, the second agent is co-administered with the first agent to produce a synergistic effect.

In certain embodiments, the first agent and one or more second agents are administered simultaneously. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and at least one second agent are prepared separately.

Example

Non-limiting disclosure and inclusion by reference

Certain compounds, compositions and methods described herein have been specifically described according to specific embodiments, but the following examples are provided merely to illustrate the compounds described herein, and are not intended to be limiting. Each of the references mentioned in this application is incorporated herein by reference in its entirety.

Example 1: Antisense inhibition of human myotonic dystrophy protein kinase (DMPK) in human skeletal muscle cells (hSKMC)

Antisense oligonucleotides targeting human DMPK nucleic acids were tested for their effect on DMPK RNA transcripts in vitro. Cultured hSKM cells at a density of 20,000 cells per well were transfected with 100 nM antisense oligonucleotides using electroporation. After about 24 hours, RNA was isolated from the cells and the level of the DMPK RNA transcript was determined by the human primer probe set RTS3164 (forward sequence AGCCTGAGCCGGGAGATG, represented herein as SEQ ID NO: 9; reverse sequence GCGTAGTTGACTGGCGAAGTT, represented herein as SEQ ID NO: 10; probe sequence. AGGCCATCCGCACGGACAACCX, represented herein as SEQ ID NO: 11) was measured by quantitative real-time PCR. DMPK RNA transcript levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of hDMPK compared to untreated control cells.

The antisense oligonucleotides of Tables 1 and 2 are 5-10-5 gapmers, wherein the gap segment contains 10 2'-deoxynucleosides, and each wing segment contains 5 2'-MOE nucleosides. Includes. The internucleoside bonds throughout each gapmer are phosphorothioate (P=S) bonds. All cytosine residues throughout each gapmer are 5-methylcytosine. The'target start site' represents the 5 most nucleosides targeted by the antisense oligonucleotide. 'Target stop site' represents the 3 most nucleosides targeted by the antisense oligonucleotide. All antisense oligonucleotides listed in Table 1 target SEQ ID NO: 1 (gene bank registration number NM_001081560.1). All antisense oligonucleotides listed in Table 2 target SEQ ID NO: 2 (complement of Genbank accession number NT_011109.15 truncated from nucleotides 18540696 to 18555106).

Several antisense oligonucleotides showed significant inhibition of human DMPK mRNA levels under the conditions specified above.

Table 1 : Inhibition of human DMPK RNA transcripts in hSKMC by 5-10-5 gapmers targeting SEQ ID NO: 1

Table 2 : Inhibition of human DMPK RNA transcripts in hSKMC by 5-10-5 gapmers targeting SEQ ID NO: 2

Antisense oligonucleotides from Tables 1 and 2 were also tested in assays with conditions similar to those described above, and mRNA levels were tested for human primer probe RTS3162 (forward sequence CGGGCCGTCCGTGTT, represented herein as SEQ ID NO: 157; reverse sequence CTTTGCACTTTGCGAACCAA, SEQ ID NO: Designated herein as 158; measured with the probe sequence CATCCTCCACGCACCCCCACCX, designated herein as SEQ ID NO: 159). The results are presented in Table 3. DMPK mRNA expression was also assessed by RTS3162 targeting the DMPK gene near 3'UTR. In order to confirm that the expression of the entire DMPK gene was suppressed, the use of a second primer probe was used.

Table 3 : Inhibition of human DMPK RNA transcripts in hSKMC by 5-10-5 gapmers measured using primer probe set RTS3162

Example 2: Design of antisense oligonucleotides targeting CUG repeats

Antisense oligonucleotides were designed that target mRNA transcripts containing multiple CUG repeats. The chemistry of these oligonucleotides as well as their sequences is shown in Table 4. The symbol designated for the sugar type is given after the base of the subscript, which is as follows: b = 2'-O-N-[2-(dimethylamino)ethyl]acetamido ribose; d = 2'-deoxyribose; e = 2'-0-methoxyethyl ribose; f = 2'-alpha-fluoro-2'-deoxyribose; g = 2'-0-2[2-(2-methoxyethoxy)ethoxy]ethyl ribose; h = 3'-fluoro-HNA; k = (S)-cEt; l = LNA (locked nucleic acid); n = 2'-0-(N-methylacetamide) ribose; o = 2'-O-dimethylaminooxyethyl (DMAOE) ribose; p = PNA; r = propylribose; And x = amino acid core. Heterocycle names are defined as standard symbols for adenine, cytosine, thymine and guanine,'mC' for 5-methylcytosine, and'K' for lysine side chain. The linker is shown after the sugar type in the subscript, which is designated by the following symbols: g = PNA-glycine total; a = amino acid; And s = thioate ester.

Table 4 : Design of antisense oligonucleotides targeting CUG repeats

Example 3: Dose-dependent antisense inhibition of human DMPK in human skeletal muscle cells

Several antisense oligonucleotides (see Example 1) showing in vitro inhibition of DMPK in hSKMC were tested at various doses. Cells were plated at a density of 20,000 cells per well and transfected with respective antisense oligonucleotides at concentrations of 1,250nM, 2,500nM, 5,000nM, 10,000nM and 20,000nM using electroporation. After about 16 hours, RNA was isolated from the cells and DMPK mRNA transcript levels were determined by quantitative real-time PCR using the primer probe set RTS3164 described above. DMPK mRNA transcript levels were normalized to total RNA content as measured by RIBOGREEN®. Results are presented in Table 5 as percent inhibition of DMPK compared to untreated control cells.

The tested antisense oligonucleotides showed a dose dependent inhibition of DMPK mRNA levels under the conditions specified above.

Table 5 : Dose dependent antisense inhibition of human DMPK in hSKMC tested with primer probe set RTS3164

Antisense oligonucleotides from Table 5 were also tested with the primer probe set RTS3162 described above. The results are presented in Table 6. DMPK mRNA expression was also assessed by RTS3162 targeting the DMPK gene near 3'UTR. In order to confirm that the expression of the entire DMPK gene was suppressed, the use of a second primer probe was used.

Table 6 : Dose dependent antisense inhibition of human DMPK in hSKMC tested with primer probe set RTS3164

Example 4: Dose-dependent antisense inhibition of human DMPK in human skeletal muscle cells

Several antisense oligonucleotides (see Example 3) showing in vitro inhibition of DMPK in hSKMC were tested at various doses. Cells were plated at a density of 20,000 cells per well and transfected with respective antisense oligonucleotides at concentrations of 1,250nM, 2,500nM, 5,000nM, 10,000nM and 20,000nM using electroporation. After about 16 hours, RNA was isolated from the cells and DMPK mRNA transcript levels were determined by quantitative real-time PCR using the primer probe set RTS3164 described above. DMPK mRNA transcript levels were normalized to total RNA content as measured by RIBOGREEN®. Results are presented in Table 7 as percent inhibition of DMPK compared to untreated control cells.

Most of the tested antisense oligonucleotides showed dose dependent inhibition of DMPK mRNA levels under the conditions specified above.

Table 7 : Dose dependent antisense inhibition of human DMPK in hSKMC tested with primer probe set RTS3164

Example 5: Dose-dependent antisense inhibition of human DMPK in human skeletal muscle cells

Several antisense oligonucleotides targeting human DMPK mRNA were designed and tested in hSKMC at various doses. Several different antisense oligonucleotides targeting human actin mRNA were designed and also tested in hSKMC at various doses. The newly designed gapmers are 2-10-2 MOE or 3-10-3 MOE gapmers. The 2-10-2 MOE gapmer is 14 nucleosides long, wherein the gap segment contains 10 2'-deoxynucleosides, and each wing segment contains 2 2'-MOE nucleosides. do. 3-10-3 MOE gapmer is 16 nucleosides long, wherein the gap segment contains 10 2'-deoxynucleosides, and each wing segment contains 3 2'-MOE nucleosides. do. The internucleoside bonds throughout each gapmer are phosphorothioate (P=S) bonds. All cytosine residues throughout each gapmer are 5-methylcytosine. The'target start site' represents the 5 most nucleosides targeted by the antisense oligonucleotide. 'Target stop site' represents the 3 most nucleosides targeted by the antisense oligonucleotide. The antisense oligonucleotides listed in Table 8 are the human DMPK genomic sequence represented herein as SEQ ID NO: 2 (complement of Genbank accession number NT_011109.15 truncated from nucleotides 18540696 to 18555106) or human actin sequence represented herein as SEQ ID NO: 801 Target (gene bank registration number NM_001100.3).

Cells were plated at a density of 20,000 cells per well and transfected with respective antisense oligonucleotides at concentrations of 1,250nM, 2,500nM, 5,000nM, 10,000nM and 20,000nM using electroporation. After about 16 hours, RNA was isolated from the cells, and DMPK mRNA transcript levels were determined by quantitative real-time PCR using the primer probe set RTS3162 described above. DMPK mRNA transcript levels were normalized to total RNA content as measured by RIBOGREEN®. Results are presented in Table 8 as percent inhibition of DMPK compared to untreated control cells. Antisense oligonucleotides were also tested under conditions similar to RTS3164. The results are presented in Table 9.

Many of the antisense oligonucleotides tested showed dose dependent inhibition of DMPK mRNA levels under the conditions specified above.

Table 8 : Dose-dependent antisense inhibition of human DMPK and human actin in hSKMC tested with primer probe set RTS3162

Table 9 : Dose dependent antisense inhibition of human DMPK in hSKMC tested with primer probe set RTS3164

Example 6: Dose response study using antisense oligonucleotides targeting human myotonic dystrophy-protein kinase (DMPK) in DM1 fibroblast cells

The mutant form of DMPK mRNA with large CUG repeats is completely transcribed and forms adenyl polymer, but remains entrapped in the nucleus (Davis et al, 1997, Proc. Natl. Acad. Sci. USA 94, 7388-7393 ). The nuclear-retained mRNA of this mutation is one of the most important pathological features of myosonic dystrophy 1 (DM1). Antisense inhibition of mutant DMPK mRNA in DM1 fibroblast cells was studied.

The DMPK gene usually has 5-37 CTG repeats within the 3'untranslated region. In myosonic dystrophy type I, this number expands significantly, which can range from 50 to more than 3,500 (Harper, Myotonic Dystrophy (Saunders, London, ed. 3, 2001); Annu. Rev. Neurosci. 29 : 259, 2006; EMBO J. 19: 4439, 2000; Curr Opin Neurol. 20: 572, 2007). DM1 fibroblast cells were plated at a density of 4,500 cells per well, and transfected with respective antisense oligonucleotides at concentrations of 9.4 nM, 18.8 nM, 37.5 nM, 75.0 nM, 150.0 nM and 300.0 nM using Cytofectin reagent. Made it. After about 16 hours, RNA was isolated from the cells and DMPK RNA transcript levels were determined by quantitative real-time PCR using the primer probe set RTS3164 described above. DMPK RNA transcript levels were normalized to total RNA content as measured by RIBOGREEN®. Results are presented in Table 10 as percent inhibition of DMPK compared to untreated control cells.

Assays with similar conditions were also performed with the above described primer probe set RTS3162 targeting the 3'-end of the DMPK transcript. Results are presented in Table 11 as percent inhibition of DMPK compared to untreated control cells.

The tested antisense oligonucleotides showed a dose dependent inhibition of DMPK mRNA levels under the conditions specified above.

Table 10 : Dose-dependent antisense inhibition of DMPK mRNA in DM1 fibroblast cells using RTS3164

Table 11 : Dose-dependent antisense inhibition of DMPK mRNA in DM1 fibroblast cells using RTS3162

Example 7: Antisense inhibition of human DMPK in human skeletal muscle cells (hSKMc)

Antisense oligonucleotides targeting human DMPK nucleic acids were tested for their effect on DMPK RNA transcripts in vitro. HSKMc cultured at a density of 20,000 cells per well was transfected with 10,000 nM antisense oligonucleotides using electroporation. After about 24 hours, RNA was isolated from the cells and DMPK transcript levels were measured by quantitative real-time PCR. DMPK RNA transcript levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of DMPK compared to untreated control cells.

The antisense oligonucleotides of Table 12 and Table 13 are 5-10-5 gapmers, wherein the gap segment contains 10 2'-deoxynucleosides, and each wing segment contains 5 2'-MOE nucleosides. Includes. The internucleoside bonds throughout each gapmer are phosphorothioate (P=S) bonds. All cytosine residues throughout each gapmer are 5-methylcytosine. 'Target start site' refers to the 5 most nucleosides that the antisense oligonucleotide targets within the human genomic gene sequence. 'Target stop site' refers to the 3'most nucleosides targeted by antisense oligonucleotides in the human genome sequence. All antisense oligonucleotides listed in Table 12 target SEQ ID NO: 1 (gene bank registration number NM_001081560.1). All antisense oligonucleotides listed in Table 13 target SEQ ID NO: 2 (complement of GenBank Accession Number NT_011109.15, truncate from nucleotides 18540696 to 18555106).

Several antisense oligonucleotides showed significant inhibition of DMPK mRNA levels under the conditions specified above.

Table 12 : Inhibition of human DMPK RNA transcripts in hSKMc by 5-10-5 gapmers targeting SEQ ID NO: 1

Table 13 : Inhibition of human DMPK RNA transcripts in hSKMc by 5-10-5 gapmers targeting SEQ ID NO: 2

Example 8: Antisense inhibition of murine DMPK in mouse primary hepatocytes

Antisense oligonucleotides targeting murine DMPK nucleic acids were tested for their effect on DMPK RNA transcripts in vitro. Cultured mouse primary hepatocytes at a density of 35,000 cells per well were transfected with 8,000 nM antisense oligonucleotides using electroporation. After about 24 hours, RNA was isolated from the cells and DMPK transcript levels were measured by quantitative real-time PCR. DMPK RNA transcript levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of DMPK compared to untreated control cells.

The antisense oligonucleotides of Tables 14, 15 and 16 are 5-10-5 gapmers, wherein the gap segment contains 10 2'-deoxynucleosides, and each wing segment contains 5 2'-MOEs. Contains nucleosides. The internucleoside bonds throughout each gapmer are phosphorothioate (P=S) bonds. All cytosine residues throughout each gapmer are 5-methylcytosine. The'murine target start site' refers to the 5'most nucleoside to which the antisense oligonucleotide is targeted in the murine gene sequence. The'murine target stop site' refers to the 3'most nucleoside to which the antisense oligonucleotide is targeted in the murine gene sequence. All antisense oligonucleotides listed in Table 12 target SEQ ID NO: 3 (Genbank accession number NT_039413.7 truncate from nucleotides 16666001 to 16681000). All antisense oligonucleotides listed in Table 13 target SEQ ID NO: 4 (gene bank registration number NM_032418.1). Antisense oligonucleotides in Table 14 are SEQ ID NO: 5 (gene bank registration number AI007148.1), SEQ ID NO: 6 (gene bank registration number AI304033.1), SEQ ID NO: 7 (gene bank registration number BC024150.1), SEQ ID NO: 8 ( Genbank registration number BC056615.1), SEQ ID NO: 793 (gene bank registration number BC075715.1), SEQ ID NO: 794 (gene bank registration number BU519245.1), SEQ ID NO: 795 (gene bank registration number CB247909.1), SEQ ID NO: 796 (gene bank registration number CX208906.1), SEQ ID NO: 797 (gene bank registration number CX732022.1), SEQ ID NO: 798 (gene bank registration number S60315.1) or SEQ ID NO: 799 (gene bank registration number S60316.1) Target. In addition, the human antisense oligonucleotide ISIS 451421 targeting SEQ ID NO: 800 (gene bank registration number NM_001081562.1) was also included in the assay, which are listed in Table 14.

The murine oligonucleotides of Tables 14, 15 and 16 can also be cross-reacted with human gene sequences. 'Mismatch' refers to the number of nucleobases at which the murine oligonucleotide mismatches the human gene sequence. The greater the complementarity between the murine oligonucleotide and the human sequence, the greater the likelihood that the murine oligonucleotide can cross-react with the human sequence. The murine oligonucleotides of Tables 14, 15 and 16 were compared with SEQ ID NO: 800 (gene bank registration number NM_001081562.1). “Human target start site” refers to the 5′ most nucleoside that the gapmer targets within the human gene sequence. “Human target stop site” refers to the 3′ most nucleoside that the gapmer targets within the human gene sequence.

Several antisense oligonucleotides tested showed significant inhibition of DMPK mRNA levels under the conditions specified above. Certain antisense oligonucleotides tested are cross-reactive with human gene sequences.

Table 14 : Inhibition of murine DMPK RNA transcripts in mouse primary hepatocytes by 5-10-5 gapmers targeting SEQ ID NO: 800

Table 15 : Inhibition of murine DMPK RNA transcripts in mouse primary hepatocytes by 5-10-5 gapmers targeting SEQ ID NO: 800

Table 16 : Inhibition of murine DMPK RNA transcripts in mouse primary hepatocytes by 5-10-5 gapmers targeting SEQ ID NOs: 5-8 and 793-799

Example 9: Dose-dependent antisense inhibition of murine DMPK in mouse primary hepatocytes

Several antisense oligonucleotides (see Example 8) showing in vitro inhibition of DMPK in mouse primary hepatocytes were tested at various doses. Cells were plated at a density of 35,000 cells per well and transfected with respective antisense oligonucleotides at concentrations of 1,000 nM, 2,000 nM, 4,000 nM, 8,000 nM and 16,000 nM using electroporation. After about 16 hours, RNA was isolated from the cells and the DMPK transcript level was determined by primer probe set RTS3181 (forward sequence GACATATGCCAAGATTGTGCACTAC, represented herein as SEQ ID NO: 771; reverse sequence CACGAATGAGGTCCTGAGCTT, represented herein as SEQ ID NO: 772; probe sequence AACACTTGTCGCTGCCGCTGGCX, It was measured by quantitative real-time PCR using (represented herein as SEQ ID NO: 773). DMPK transcript levels were normalized to total RNA content as measured by RIBOGREEN®. Results are presented in Table 17 as percent inhibition of DMPK compared to untreated control cells.

Most of the tested antisense oligonucleotides showed dose dependent inhibition of DMPK mRNA levels under the conditions specified above.

Table 17 : Dose-dependent antisense inhibition of murine DMPK in mouse primary hepatocytes

Example 10: Antisense inhibition of human alpha1 skeletal actin in HepG2 cells

Antisense oligonucleotides targeting human alpha1 scaffold actin nucleic acids, a gene that can have extended CTG repeats that can cause symptoms of DM1 when inserted into a mouse model, are used in vitro against alpha1 actin RNA transcripts. Tested for effectiveness. HepG2 cells cultured at a density of 20,000 cells per well were transfected with 10,000 nM antisense oligonucleotides using electroporation. After about 24 hours, RNA was isolated from the cells, and alpha1 actin RNA transcript levels were measured by quantitative real-time PCR. Alpha1 actin RNA transcript levels were adjusted according to the total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of alpha1 actin compared to untreated control cells.

The antisense oligonucleotides of Table 18 are 5-10-5 gapmers, wherein the gap segment contains 10 2'-deoxynucleosides, and each wing segment contains 5 2'-MOE nucleosides. . The internucleoside bonds throughout each gapmer are phosphorothioate (P=S) bonds. All cytosine residues throughout each gapmer are 5-methylcytosine. The'target start site' represents the 5 most nucleosides targeted by the antisense oligonucleotide. 'Target stop site' represents the 3 most nucleosides targeted by the antisense oligonucleotide. All antisense oligonucleotides listed in Table 18 target SEQ ID NO: 801 (gene bank registration number NM_001100.3).

The tested antisense oligonucleotide sequences showed dose dependent inhibition of alpha1 actin mRNA levels under the conditions specified above.

Table 18 : Inhibition of human alpha1 actin RNA transcripts in HepG2 cells by 5-10-5 gapmers targeting SEQ ID NO: 801

Example 11: Dose-dependent antisense inhibition of human alpha1 actin in HepG2 cells

Several antisense oligonucleotides (see Example 8) showing in vitro inhibition of alpha1 actin in HepG2 cells were tested at various doses. Cells were plated at a density of 20,000 cells per well and transfected with respective antisense oligonucleotides at concentrations of 625 nM, 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM using electroporation. After about 16 hours, RNA was isolated from cells and the alpha1 actin RNA transcript level was determined by primer probe set RTS3154 (forward CCACCGCAAATGCTTCTAGAC, represented herein as SEQ ID NO: 785; reverse CCCCCCCATTGAGAAGATTC, represented herein as SEQ ID NO: 786; probe sequence CTCCACCTCCAGCACGCGACTTCTX , Represented herein as SEQ ID NO: 787) by quantitative real-time PCR. Alpha1 actin RNA transcript levels were normalized according to total RNA content as measured by RIBOGREEN®. Results are presented in Table 19 as percent inhibition of alpha1 actin relative to untreated control cells.

Several antisense oligonucleotides showed dose dependent inhibition of alpha1 actin mRNA levels under the conditions specified above.

Table 19 : Dose-dependent antisense inhibition of human alpha1 actin in HepG2 cells

Example 12: In vivo antisense inhibition of human alpha1 actin by intramuscular administration in transgenic mice

In order to test the effect of antisense inhibition for the treatment of muscular dystrophy, an appropriate mouse model was needed. The HSA ^LR mouse model is an established model for DM1 (Mankodi, A. et al. Science. 289: 1769, 2000). The mouse has a human skeletal actin (hACTA1) transgene with 220 CTG repeats inserted into the 3'UTR of the gene. The hACTA1-CUGexp transcript accumulates into the nuclear foci in skeletal muscle, which causes dystonia similar to that of human DM1 (Mankodi, A. et al. Mol. Cell 10: 35, 2002; Lin, X. et al. Hum. Mol. Genet. 15: 2087, 2006). ^{Therefore, it was expected that the improvement of DM1 symptoms in HSA LR} mice by antisense inhibition of the hACTA1 transgene predicted the improvement of similar symptoms in human patients by antisense inhibition of the DMPK transcript.

HSA (human skeletal actin) ^LR (long repeat) DM1 mice were generated by insertion of a transgene into FVB/N mice with 250 CUG repeats in the 3'UTR of human skeletal actin. The transgene is expressed in mice as CUG repeat RNA that is retained in the nucleus, resulting in the formation of a nuclear inclusion or focal point similar to that observed in human tissue samples from patients with muscular dystrophy (DM1).

ISIS 190403 and ISIS 445238 (see Example 11), which showed statistically significant dose dependent inhibition in vitro, were evaluated for their ability to reduce human alpha1 actin RNA transcripts in vivo.

process

HSA ^LR mice were maintained on a 12-hour light/dark cycle, and were fed unrestrictedly with normal Purina mouse food. Animals were acclimated to the new environment for at least 7 days in the study facility prior to initiation of the experiment. Antisense oligonucleotides (ASO) were prepared in PBS and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Mice were divided into two treatment groups. Two groups were administered ISIS 190403 or ISIS 445238 by direct intramuscular injection at a dose of 0.8 nM to the anterior tibial muscle on one side. PBS was administered as a single dose of intramuscular injection to the opposite anterior tibial muscle of each mouse. PBS injected muscles served as a control.

Inhibition of alpha1 actin RNA

24 hours after the final administration, the animals were sacrificed and the tissue was separated from the anterior tibial muscle on both sides. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 20, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

The above results indicate that treatment with ISIS 190403 and ISIS 445238 resulted in inhibition of alpha1 actin RNA levels in mice.

Table 20 : Percent inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Example 13: Dose-dependent antisense inhibition of human alpha1 actin by intramuscular administration in transgenic mice

ISIS 445236 (see Example 11), which showed statistically significant dose dependent inhibition in vitro, was evaluated for its ability to reduce human alpha1 actin RNA transcripts in vivo.

process

HSA ^LR mice were maintained on a 12-hour light/dark cycle, and were fed unrestrictedly with normal Purina mouse food. Animals were acclimated to the new environment for at least 7 days in the study facility prior to initiation of the experiment. Antisense oligonucleotides (ASO) were prepared in PBS and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Mice were divided into 3 treatment groups. The group was administered ISIS 445236 by direct intramuscular injection at a dose of 0.2 nM, 0.4 nM or 0.8 nM to the anterior tibial muscle of one side. PBS was administered as a single dose of intramuscular injection to the opposite anterior tibial muscle of each mouse. PBS injected muscles served as a control.

Inhibition of alpha1 actin RNA

24 hours after the final administration, the animals were sacrificed and the tissue was separated from the anterior tibial muscle on both sides. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 21, treatment with ISIS 445236 reduced human alpha1 actin RNA transcript expression at all doses. Results are expressed as percent inhibition of alpha1 actin transcript compared to control.

The results indicate that treatment with ISIS 445236 resulted in significant inhibition of alpha1 actin mRNA levels under the conditions specified above.

Table 21 : Inhibition of human alpha1 actin RNA transcripts by ISIS 445236 in ^{HSA LR mice}

Evaluation of muscle tone by electromyography

Muscular dystonia refers to a recurrent action potential due to delayed relaxation of muscle fibers. This phenomenon is observed in ^{HSA LR} mice as well as patients with dystrophic dystrophy. When an EMG needle is inserted into the dystonia muscle, electrical activity is usually extended to a few seconds after the insertion activity is stopped. The frequency of the dystonia discharge ranges from 50 to 100 impulses per second.

Muscular dystonia was measured via electromyography and graded in the following manner: Grade 0 indicates that dystonia was not induced by any needle insertion (0%); Grade 1 indicates that muscle tone was induced by less than 50% needle insertion; Grade 2 indicates that muscle dystonia was induced by at least 50% needle insertion; Grade 3 indicates that muscle tone was induced by 100% needle insertion.

Before electromyography, mice were anesthetized using a cocktail of 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg acepromazine intraperitoneally. Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 22 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 445236.

Table 22 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Correction of alternative splicing

In the DM1/HSA ^LR mouse model, accumulation of expanded CUG RNA in the nucleus results in sequestration of poly(CUG)-binding proteins such as Muscleblind-like 1 (MBLN1) (Miller, JW et al. EMBO J. 19: 4439, 2000). The splicing factor MBNL1, which regulates alternative splicing of the Serca1 gene, is isolated within the expanded CUG foci. This triggers dysregulation of alternative splicing of the gene. In order to evaluate the effect of antisense inhibition of human alpha1 actin on the alternative splicing, total RNA was prepared according to the manufacturer's instructions using the RNeasy Lipid Tissue Mini Kit (Qiagen). Purified from. RT-PCR was performed with Superscript III One-Step RT-PCR System and Platinum Taq Polymerase (Invitrogen) using gene-specific primers for cDNA synthesis and PCR amplification. Forward and reverse primers for Serca-1 are described by Bennett and Swayze (Annu. Rev. Pharmacol. 2010; 50: 259-93). The PCR product was separated on an agarose gel, stained with SybrGreen I Nucleic Acid Gel Stain (Invitrogen), and imaged using a Fujifilm LAS-3000 Intelligent Dark Box.

Serca1's PCR product of the splice in the PBS control group was characterized by the 22 exons excluded as a result of the above adjustment of MBLN1. Treatment with ISIS 445236 resulted in exon 22 inclusion and normalization of alternative splicing of the Serca1 gene in the anterior tibialis, calf and quadriceps muscles.

Thus, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, indicating that treatment with antisense oligonucleotides reduced the accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects for MBLN1 sequestration, allowing normal splicing to occur.

Example 14: In vivo antisense inhibition of human alpha1 actin by subcutaneous administration in transgenic mice

ISIS 190403, ISIS 445236 and ISIS 445238 were evaluated for their ability to reduce human alpha1 actin RNA transcripts in vivo.

process

HSA ^LR mice were maintained on a 12-hour light/dark cycle, and were fed unrestrictedly with normal Purina mouse food. Animals were acclimated to the new environment for at least 7 days in the study facility prior to initiation of the experiment. Antisense oligonucleotides (ASO) were prepared in PBS and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Mice were divided into 4 treatment groups. The first three groups were administered ISIS 190403, ISIS 445236 or ISIS 445238 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The fourth group was administered PBS by subcutaneous injection twice a week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Inhibition of alpha1 actin RNA

24 hours after the final administration, animals were sacrificed, and tissues were separated from the quadriceps muscle (left and right), calf muscle (left and right) and anterior tibialis muscle (left and right). RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 23, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. Results are expressed as percent inhibition of alpha1 actin transcript compared to control.

Both ISIS 445236 and ISIS 445238 showed significant inhibition of alpha1 actin mRNA levels under the conditions specified above.

Table 23 : Percent inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Fluorescence in situ hybridization of alpha1 actin in muscle

Frozen muscle tissue sections were fixed in a 3% paraformaldehyde solution in fresh PBS for 15-20 minutes, then rinsed twice with PBS for 5 minutes. After the nuclei were permeabilized with 0.5% Triton X-100 for 5 minutes, the tissues were blocked with normal goat serum for 30 minutes. Sections were incubated with 2'-O-methyl RNA targeting 5'-labeled alpha1 actin with Texas Red (Integrated DNA Technologies). Sections were counterstained with DAPI to label the nuclei. The section was placed on a standard fluorescence microscope and observed. Images were acquired by Metavue software, and deconvolution was achieved by Autoquant software.

All muscle tissue sections from mice treated with ISIS 445236 and ISIS 445238 showed a reduced fluorescence intensity of the alpha1 actin signal in the ribonucleic foci, which is an antisense inhibition of human alpha1 actin mRNA and a reduction of RNA in the nuclear foci. Represents.

Evaluation of muscle tone by electromyography

Muscular dystonia refers to a recurrent action potential due to delayed relaxation of muscle fibers. This phenomenon is observed in ^{HSA LR} mice as well as patients with dystrophic dystrophy. When an EMG needle is inserted into the dystonia muscle, electrical activity is usually extended to a few seconds after the insertion activity is stopped. The frequency of the dystonia discharge ranges from 50 to 100 impulses per second.

Muscular dystonia was measured via electromyography and graded in the following manner: Grade 0 indicates that dystonia was not induced by any needle insertion (0%); Grade 1 indicates that muscle tone was induced by less than 50% needle insertion; Grade 2 indicates that muscle dystonia was induced by at least 50% needle insertion; Grade 3 indicates that muscle tone was induced by 100% needle insertion.

Before electromyography, mice were anesthetized using 100 mg/kg ketamine, 10 mg/kg xylazine and 3 mg/kg acepromazine or 250 mg/kg 2,2,2-tribromoethanol intraperitoneally. Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 24 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 445236 and ISIS 445238.

Table 24 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Correction of alternative splicing

The splicing factor MBNL1, which regulates Serca1 splicing, m-Titin splicing, CIC-1 chloride channel gene ( Clcn1 ) splicing, and Zasp splicing, is isolated within the extended CUG foci. MBNL1 sequestration triggers dysregulated splicing in each of these genes. To evaluate the effect of antisense inhibition of human alpha1 actin on splicing, total RNA was purified from the anterior tibialis, calf and quadriceps muscles as described in Example 13, and RT-PCR was performed. Forward and reverse primers for Serca-1 , m-Titin , Clcn1 and ZASP are described in Bennett and Swayze, Annu. Rev. Pharmacol. 2010; 50: 259-93.

In PBS treated HSA ^LR mice, Serca1 splicing is dysregulated as evidenced by exon 22 exclusion. Treatment with ISIS 445236 and ISIS 445238, respectively, resulted in normalization of exon 22 inclusion and alternative splicing of the Serca1 gene in the anterior tibialis, calf and quadriceps muscles.

In PBS treated HSA ^LR mice, m-Titin splicing is dysregulated as evidenced by exon 5 inclusion. Treatment with ISIS 445236 and ISIS 445238, respectively, resulted in the normalization of skipping of exon 5 and alternative splicing of the m-Titin gene in the anterior tibialis, calf and quadriceps muscles.

In PBS treated HSA ^LR mice, Clcn1 splicing is dysregulated as evidenced by exon 7a inclusion. Treatment with ISIS 445236 and ISIS 445238, respectively, resulted in the normalization of skipping of exon 7a and alternative splicing of the Clcn1 gene in the anterior tibialis, calf and quadriceps muscles.

In PBS treated HSA ^LR mice, Zasp splicing is dysregulated as evidenced by exon 11 inclusion. Treatment with ISIS 445236 and ISIS 445238, respectively, resulted in the normalization of skipping of exon 11 and alternative splicing of the Zasp gene in the anterior tibialis, calf and quadriceps muscles.

Thus, antisense inhibition of alpha1 actin corrected Serca1, m-Titin, Clcn1 and Zasp splicing dysregulation, indicating that treatment with antisense oligonucleotides reduced the accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects for MBLN1 sequestration, allowing normal splicing to occur.

Example 15: In vivo antisense inhibition of human alpha1 actin in transgenic mice

Antisense inhibition of human alpha1 actin RNA transcripts by ISIS 445236 and ISIS 445238 against myotonia in HSA ^{LR mice was further evaluated.}

process

HSA ^LR mice were divided into three treatment groups. The first two groups were administered ISIS 445236 or ISIS 445238 by subcutaneous injection at a dose of 25 mg/kg twice per week for 2 weeks. The third group was administered PBS by subcutaneous injection twice a week for 2 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Inhibition of alpha1 actin RNA

24 hours after the final administration, the animals were sacrificed, and tissues were separated from the quadriceps muscle, calf muscle and anterior tibialis muscle. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 25, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

Both ISIS 445236 and ISIS 445238 showed significant inhibition of alpha1 actin mRNA levels under the conditions specified above.

Table 25 : Percent inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 26 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 445236 and ISIS 445238.

Table 26 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Correction of alternative splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1 , total RNA purified from the anterior tibialis calf and quadriceps muscles was analyzed with a procedure similar to that described in Example 13.

In PBS treated HSA ^LR mice, Serca1 splicing is dysregulated as evidenced by exon 22 exclusion as a result of MBLN1 dysregulation. Treatment with ISIS 445236 and ISIS 445238 respectively resulted in near complete inclusion in the anterior tibialis and quadriceps muscles and normalization of alternative splicing of exon 22 of the Serca1 gene.

Thus, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, indicating that treatment with antisense oligonucleotides reduced the accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects for MBLN1 sequestration, allowing normal splicing to occur.

Example 16: Dose-dependent antisense inhibition of human alpha1 actin in transgenic mice

Dose-dependent inhibition of human alpha1 actin RNA transcripts by ISIS 445236 and ISIS 445238 on myotonia in HSA ^{LR mice was evaluated.}

process

HSA ^LR mice were injected subcutaneously with ISIS 445236 or ISIS 445238 at doses of 2.5 mg/kg, 8.5 mg/kg or 25.0 mg/kg twice per week for 4 weeks. The control group was administered PBS by subcutaneous injection twice per week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Inhibition of alpha1 actin RNA

24 hours after the final administration, animals were sacrificed, and tissues were separated from the quadriceps muscle (Quad), calf muscle (Gastroc) and anterior tibialis muscle (TA). RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 27, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

Both antisense oligonucleotides showed dose-dependent inhibition of alpha1 actin mRNA levels in the quadriceps muscle, calf muscle and anterior tibialis muscle under the conditions specified above.

Table 27 : Dose-dependent inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right calf muscles (Gastroc), left and right anterior tibialis (TA) muscles, and lumbar lateral vertebrae muscles were tested with a 30 gauge concentric needle electrode and minimum for each muscle. It was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) using 10 needle insertions. Data are presented in Table 28 as the average dystonia grade observed in 4 mice in each group, indicating a significant dose-dependent reduction in dystonia in mice treated with ISIS 445236 and ISIS 445238.

Table 28 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Correction of alternative splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1 , total RNA purified from the anterior tibialis calf and quadriceps muscles was analyzed with a procedure similar to that described in Example 13.

In PBS treated HSA ^LR mice, Serca1 splicing is dysregulated as evidenced by exon 22 exclusion as a result of MBLN1 dysregulation. Treatment with ISIS 445236 or ISIS 445238 at doses of 8.5 mg/kg or 25.0 mg/kg twice a week (or 17.0 mg/kg/week and 50.0 mg/kg/week) complete inclusion in all three muscle types. And normalization of alternative splicing of exon 22 of the Serca1 gene.

Thus, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, indicating that treatment with antisense oligonucleotides reduced the accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects for MBLN1 sequestration, allowing normal splicing to occur.

Example 17: In vivo antisense inhibition by oligonucleotides targeting the HSA coding region of human alpha1 actin in transgenic mice

Antisense inhibition of human alpha 1 actin RNA transcripts by ISIS 190401 (5'-GCGGTCAGCGATCCCAGGGT-3' (SEQ ID NO: 788), target start site 1028 of SEQ ID NO: 1) against myotonia in HSA ^{LR mice was evaluated.}

process

HSA ^LR mice were administered ISIS 190401 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The control group was administered PBS by subcutaneous injection twice per week for 2 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Inhibition of alpha1 actin RNA

24 hours after the final administration, the animals were sacrificed, and tissues were separated from the quadriceps muscle, calf muscle and anterior tibialis muscle. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 29, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

Treatment with ISIS 190401 resulted in significant inhibition of alpha1 actin mRNA levels in the quadriceps muscle, calf muscle and anterior tibialis muscle under the conditions specified above.

Table 29 : Antisense inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 30 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 190401.

Table 30 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Correction of alternative splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1 , total RNA purified from the anterior tibialis calf and quadriceps muscles was analyzed with a procedure similar to that described in Example 13.

In PBS treated HSA ^LR mice, Serca1 splicing is dysregulated as evidenced by exon 22 exclusion as a result of MBLN1 dysregulation. Treatment with ISIS 190401 resulted in complete inclusion in all three muscle types and normalization of alternative splicing of exon 22 of the Serca1 gene.

Thus, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, indicating that treatment with antisense oligonucleotides reduced the accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear focal point corrects for MBLN1 sequestration, allowing normal splicing to occur.

Example 18: Duration of action of antisense inhibition by oligonucleotides targeting human alpha1 actin in transgenic mice

The duration of action of antisense inhibition of human alpha1 actin RNA transcripts by ISIS 445236 in ^{HSA LR mice was evaluated.}

process

HSA ^LR mice were administered ISIS 445236 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The control group was administered PBS by subcutaneous injection twice per week for 2 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group. Mice were analyzed for 6 weeks after administration of the last dose.

Inhibition of alpha1 actin RNA

Six weeks after the final administration, the animals were sacrificed, and tissues were separated from the quadriceps muscle, calf muscle and anterior tibialis muscle. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 31, treatment with ISIS 445236 reduced human alpha1 actin RNA transcript expression, and this effect persisted for at least 6 weeks. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

Treatment with ISIS 445236 resulted in significant inhibition of alpha1 actin mRNA levels in the quadriceps muscle, calf muscle and anterior tibialis muscle under the conditions specified above.

Table 31 : Antisense inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 32 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 445236. Thus, the effect of antisense inhibition of alpha actin by ISIS 445236 lasted for at least 6 weeks.

Table 32 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Example 19: In vivo effect of antisense inhibition of mRNA with CUG repeats by intramuscular administration in transgenic mice

The effect of antisense inhibition of mRNA transcripts containing multiple CUG repeats was evaluated on muscle tone in HSA ^{LR mice.} Targeting the CUG repeats, three antisense oligonucleotides of varying length were assayed for their effect in inhibiting dystonia in mice. ISIS 444745 (AGCAGCAGCAGCAGCAGCAGCAGCA (SEQ ID NO: 789) is a 25 nucleotide long homogeneous 2'-O-methoxyethyl oligonucleotide with a phosphorothioate backbone. ISIS 444746 (AGCAGCAGCAGCAGCAGCAG (SEQ ID NO: 790) is a phosphorothioate backbone). It is a uniform 2'-O-methoxyethyl oligonucleotide of 20 nucleotides long with a backbone.ISIS 444749 (GCAGCAGCAGCAGCA (SEQ ID NO: 791) is a uniform 2'-O of 15 nucleotides long with a phosphorothioate backbone. -Methoxyethyl oligonucleotide ISIS 445236 was included in the assay as a positive control.

process

HSA ^LR mice were divided into three treatment groups. The group was administered ISIS 444745, ISIS 444746 or ISIS 444749 by direct intramuscular injection at a dose of 0.4 nM to the anterior tibial muscle. PBS was administered as a single dose of intramuscular injection to the opposite anterior tibial muscle of each mouse. PBS injected muscles served as a control.

Inhibition of alpha1 actin RNA

24 hours after the final administration, animals were sacrificed and tissues were separated from the anterior tibialis (left and right). RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 33, only treatment with ISIS 444745 reduced human alpha1 actin RNA transcript expression. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

Table 33 : Percent inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Example 20: In vivo dose-dependent inhibition of mRNA with CUG repeats by intramuscular administration in transgenic mice

ISIS 444745 and ISIS 444746 were further evaluated for their ability to reduce human alpha1 actin mRNA in vivo.

process

HSA ^LR mice were maintained on a 12-hour light/dark cycle, and were fed unrestrictedly with normal Purina mouse food. Animals were acclimated to the new environment for at least 7 days in the study facility prior to initiation of the experiment. Antisense oligonucleotides (ASO) were prepared in PBS and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Mice were divided into 6 treatment groups. In three groups, ISIS 444745 was administered by direct intramuscular injection at a dose of 0.2 nM, 0.5 nM or 1.0 nM to the anterior tibial muscle on one side. In another 3 groups, ISIS 444746 was administered by direct intramuscular injection at a dose of 0.2 nM, 0.5 nM or 1.0 nM to the anterior tibial muscle on one side. PBS was administered as a single dose of intramuscular injection to the opposite anterior tibial muscle of each mouse. PBS injected muscles served as a control for the corresponding muscles treated with ISIS oligonucleotides.

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 34 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 444745 or ISIS 444746. The effect of antisense inhibition of alpha actin by ISIS 444745 and 444746 lasted for at least 6 weeks.

Table 34 : Dose-dependent reduction of muscle tone in the muscles of antisense oligonucleotide-treated HSA ^{LR mice}

Example 21: In vivo effect of antisense inhibition of mRNA with CUG repeats by subcutaneous administration in transgenic mice

The effect of antisense inhibition of mRNA transcripts containing multiple CUG repeats was evaluated on muscle tone in HSA ^{LR mice.} ISIS 445236 was included in the assay as a positive control.

process

HSA ^LR mice were divided into 5 treatment groups. The first three groups were administered ISIS 444745, ISIS 444746 or ISIS 444749 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The fourth group was administered PBS by subcutaneous injection twice a week for 4 weeks. The fifth group was administered ISIS 445236 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 35 as the average dystonia grade observed in 4 mice in each group.

Treatment with ISIS 445236 resulted in a significant reduction in dystonia. Treatment with ISIS 444745 and ISIS 444746 also resulted in reduced myotonia in some of the tissues tested.

Table 35 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Example 22: Long CUG repeat mRNA by subcutaneous administration in transgenic mice (HSA ^LR Mouse) and short CUG repeats (HSA ^SR Mice) dose-dependent inhibition

Dose-dependent inhibition of mRNA transcripts containing long CUG repeats (HSA ^LR mice) and short CUG repeats (HSA ^{SR mice) was evaluated.} HSA-short repeat (HSA ^SR ) mice express the same transgene as ^{HSA LR} mice, except that 5 CUG repeats instead of 250 were inserted into the 3'UTR. HSA ^SR mice do not have dystonia, splicing changes, or any other observable dystonia phenotype. ISIS 445236 was used for this assay.

process

HSA ^LR mice were divided into 4 treatment groups. The first three groups were administered ISIS 445236 by subcutaneous injection at a dose of 2.5 mg/kg, 8.5 mg/kg or 25.0 mg/kg twice per week for 4 weeks. The fourth group was administered PBS by subcutaneous injection twice a week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group. HSA ^SR mice were also divided into 4 groups and treated similarly.

Inhibition of alpha1 actin RNA

24 hours after the final administration, animals were sacrificed, and tissues were separated from the quadriceps muscle (left and right), calf muscle (left and right) and anterior tibialis muscle (left and right). RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. Results are presented in Tables 36 and 37, which are expressed as percent inhibition of alpha1 actin transcript compared to control. A greater inhibition of nuclear retained long repeats in the muscles of ^{HSA LR} mice was achieved compared to non-nuclear retained short repeats in the muscles of ^{HSA SR mice.}

Table 36 : Percent inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Table 37 : Percent inhibition of human alpha1 actin RNA transcripts in ^{HSA SR mice}

Example 23: In vivo antisense inhibition of human DMPK in transgenic mice

LC15 mouse strain A is a transgenic mouse containing the whole human DMPK 3'UTR (developed by Wheeler et al, University of Rochester). The mice are the second generation of mice backcrossed in the FVB background. The transgene is expressed in mice as CUG repeat RNA retained in the nucleus, forming intranuclear inclusion bodies or foci similar to those observed in human tissue samples from patients with myotonic dystrophy (DM1). There are 350-400 CUG repeats in the DMPK transgene. These mice show early signs of DM1 and do not show any dystonia in the muscle tissue.

ISIS 445569, ISIS 444404, ISIS 444436 and ISIS 473810 (see Example 5), which showed statistically significant dose dependent inhibition in vitro, were evaluated for their ability to reduce human DMPK RNA transcripts in vivo.

process

LC15 line A mice were maintained on a 12-hour light/dark cycle, and were fed unrestrictedly with normal Purina mouse food. Animals were acclimated to the new environment for at least 7 days in the study facility prior to initiation of the experiment. Antisense oligonucleotides (ASO) were prepared in PBS and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Mice were divided into 5 treatment groups. The first three groups were administered ISIS 445569, ISIS 444404 or ISIS 444436 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The fourth group was administered ISIS 473810 by subcutaneous injection at a dose of 12.5 mg/kg twice per week for 4 weeks. The fifth group was administered PBS by subcutaneous injection twice a week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Inhibition of DMPK RNA

24 hours after the final administration, the animals were sacrificed and the tissue was separated from the quadriceps muscle. RNA was isolated for real-time PCR analysis of DMPK and normalized to 18s RNA. As shown in Table 38, treatment with antisense oligonucleotides reduced human DMPK RNA transcript expression. Results are expressed as percent inhibition of DMPK transcript compared to PBS control.

Table 38 : Antisense inhibition of human DMPK RNA transcripts in LC15 mice

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Since LC15 mice did not have dystonia, the control and treatment groups did not show any dystonia in any of the muscles tested.

Example 24: In vivo antisense inhibition of human DMPK in transgenic mice

LC15 mouse strain D is a transgenic mouse containing the whole human DMPK 3'UTR (developed by Wheeler et al, University of Rochester). The mice are the third generation of mice backcrossed in the FVB background. The transgene is expressed in mice as CUG repeat RNA maintained in the nucleus, forming an intranuclear inclusion or focal point similar to that observed in human tissue samples from patients with muscular dystrophy (DM1). There are 350-400 CUG repeats in the DMPK transgene. These mice show early signs of DM1 and do not show any dystonia in the muscle tissue.

ISIS 445569, ISIS 444404, ISIS 444436 and ISIS 473810 were further evaluated for their ability to reduce human DMPK RNA transcripts in vivo.

process

LC15 line D mice were maintained on a 12-hour light/dark cycle, and were fed unrestrictedly with normal Purina mouse food. Animals were acclimated to the new environment for at least 7 days in the study facility prior to initiation of the experiment. Antisense oligonucleotides (ASO) were prepared in PBS and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Mice were divided into 6 treatment groups. The first three groups were administered ISIS 445569, ISIS 444404 or ISIS 444436 by subcutaneous injection at a dose of 25.00 mg/kg twice per week for 4 weeks. The fourth group was administered ISIS 473810 by subcutaneous injection at a dose of 12.50 mg/kg twice per week for 4 weeks. The fifth group was administered ISIS 473810 by subcutaneous injection at a dose of 6.25 mg/kg twice per week for 4 weeks. The sixth group was administered PBS by subcutaneous injection twice a week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Inhibition of DMPK RNA

24 hours after the final administration, the animals were sacrificed and the tissue was separated from the quadriceps muscle. RNA was isolated for real-time PCR analysis of DMPK and normalized to 18s RNA. As shown in Table 39, treatment with antisense oligonucleotides reduced human DMPK RNA transcript expression. Results are expressed as percent inhibition of DMPK transcript compared to PBS control.

The above results indicate that treatment with antisense oligonucleotides caused inhibition of DMPK mRNA in mice.

Table 39 : Antisense inhibition of human DMPK RNA transcripts in LC15 mice

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Since LC15 mice did not have dystonia, the control and treatment groups did not show any dystonia in any of the muscles tested.

Example 25: In vivo antisense inhibition of human DMPK in SXL transgenic mouse model

Using hDMPK-targeted ASO, 444401 and 299471 target knockdown in the soleus muscle were measured in SXL mice. SXL mice are transgenic for the entire DMPK gene and promoter, and contain 1000 CUG repeat sequences in the 3'UTR of the DMPK gene. Mice were dosed with 50 mg/kg twice per week for 4 weeks (n=3 mice per group, n=2 except saline injected control group). The results of the Taqman assay showed that treatment with ISISI 444401 or ISIS 299471 significantly reduced mut-hDMPK mRNA levels, but slightly affected the endogenous mouse Dmpk mRNA levels.

Thus, ISIS 444401 and ISIS 299471 selectively target human DMPK mRNA transcripts.

Example 26: Duration of action of antisense inhibition by oligonucleotides targeting human alpha1 actin in transgenic mice

The duration of action of antisense inhibition of human alpha1 actin RNA transcripts by ISIS 190401 in ^{HSA LR mice was evaluated.}

process

HSA ^LR mice were administered ISIS 190401 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The control group was administered PBS by subcutaneous injection twice per week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group. Mice were analyzed for 15 weeks after administration of the last dose.

Inhibition of alpha1 actin RNA

15 weeks after the final administration, animals were sacrificed, and tissues were separated from the quadriceps muscle, calf muscle and anterior tibialis muscle. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As shown in Table 40, treatment with ISIS 190401 reduced human alpha1 actin RNA transcript expression, and this effect lasted for at least 15 weeks. Results are expressed as percent inhibition of alpha1 actin transcript compared to PBS control.

Treatment with ISIS 190401 resulted in significant inhibition of alpha1 actin mRNA levels under the conditions specified above.

Table 40 : Antisense inhibition of human alpha1 actin RNA transcripts in ^{HSA LR mice}

Evaluation of muscle tone by electromyography

Electromyography of the left and right quadriceps, left and right rectus muscles, left and right anterior tibialis muscles, and lumbar vertebral muscles were previously performed using a 30 gauge concentric needle electrode and at least 10 needle insertions for each muscle. It was performed as described (Kanadia et al, 2003, Science, 302: 1978-1980). Data are presented in Table 41 as the average dystonia grade observed in 4 mice in each group, indicating a significant reduction in dystonia in mice treated with ISIS 190401. Thus, the effect of antisense inhibition of alpha actin by ISIS 190401 lasted for at least 15 weeks.

Table 41 : Mean reduction of dystonia in various muscles ^{of HSA LR} mice treated with antisense oligonucleotides

Correction of alternative splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1 , total RNA purified from the anterior tibialis calf and quadriceps muscles was analyzed with a procedure similar to that described in Example 13.

In PBS treated HSA ^LR mice, Serca1 splicing is dysregulated as evidenced by exon 22 exclusion. Treatment with ISIS 190401 resulted in complete inclusion in all three muscle types and normalization of alternative splicing of exon 22 of the Serca1 gene, which persisted after 15 weeks.

Thus, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, indicating that treatment with antisense oligonucleotides reduced the accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects for MBLN1 sequestration, allowing normal splicing to occur.

Example 27: Microarray analysis of the transcriptomic effect of antisense inhibition of human actin

Expression of actin mRNA with extended CUG repeats leads to extensive remodeling of muscle transcripts. To evaluate the overall transcriptome effect of ISIS 190401 and ISIS 445236, microarray analysis was used ^{in HSA LR mice.}

process

HSA ^LR mice were administered ISIS 190401 or ISIS 445236 by subcutaneous injection at a dose of 25 mg/kg twice per week for 4 weeks. The control group was administered PBS by subcutaneous injection twice per week for 4 weeks. The PBS-injected group served as a control compared to the oligonucleotide-treated group.

Transcript analysis by microarray

RNA was isolated from the quadriceps muscle of wild type or HSA ^{LR mice.} RNA integrity was confirmed using an Agilent Bioanalyzer (RNA integrity number> 7.5). RNA was treated with complementary RNA (cRNA) and hybridized on microbeads using MouseRef-8 v2.0 Expression BeadChip Kits (Illumina, San Diego) according to the manufacturer's recommendations. Image data was quantified using BeadStudio software (Illumina). The signal intensity was standardized by quantiles. Row-specific offset was used to avoid any value less than 2 before normalization. Six CUG, UGC or GCU repeats to exclude the possibility that extended repeats in the hybridization mixture (CAG repeats in cRNA derived from CUG repeats in mRNA) may cross-hybridize with repeat sequences in the probe. Data from all probe sets having the above nucleotides were deleted. In order to exclude genes whose expression is not readily quantified in the array, probes showing P values for probability of detection less than 0.1 were deleted from all samples. Comparisons between groups were summarized and ranked by mean expression level and fold change of t test. Key component analysis for wild-type samples, ISIS oligonucleotide-treated samples and PBS-treated microarray samples (Levin et al . In Antisense Drug Technology: Principles, Strategies, and Applications , ST Crooke, Ed. (CRC Press, Boca Raton) , 2008), pp 183-215; Geary et al. Drug Metab. Dispos . 2003; 31: 1419-28) using software package R (Butler et al. Diabetes . 2002; 51: 1028-34) I did. The main component allows the capture of most of the expression changes of each sample within three dimensions. The first three major components of each sample were constructed.

Analysis of the major components of untreated wild-type and HSA ^LR mice revealed the isolation of ^{HSA LR} away from a broadly clustered population of wild-type mice. In contrast, antisense oligonucleotide treated HSA ^LR mice clustered more closely to wild-type mice, suggesting a trend of overall transcript normalization. A comparison of HSA ^LR transgenic mice and wild-type mice confirmed 93 transcripts whose expression levels were changed by more than two times (P <0.0001) as shown in Table 42 below. The degree of dysregulation for the transcript was reduced or normalized for antisense oligonucleotides (88% of dysregulated transcripts responded to ISIS 445236, P <0.05 for ISIS 445236, versus 90% for ISIS 190401. Reacted PBS control).

In order to consider transcripts with off-target knockdown, all transcripts with ^{reduced expression in antisense oligonucleotide-treated HSA LR} mice were identified (reduction of more than 2 fold by any one oligonucleotide. , P <0.0001, n = 41 transcript). All transcripts downregulated by this criterion showed upregulation ^{in HSA LR mice.} The only exception, collagen 6 alpha2, is unlikely to arise from off-target cleavage, as the collagen 6 alpha2 is downregulated by two antisense oligonucleotides with non-overlapping sequences.

These results indicate that treatment with antisense oligonucleotides for 4 weeks resulted in overall improvement of the muscle transcriptome without any evidence of off-target effects.

Table 42 : ^{Comparison of 93 transcripts identified in HSA LR} transgenic mice and wild-type mice

SEQUENCE LISTING <110> C. Frank Bennett Susan M. Freier Robert A. MacLeod Sanjay K. Pandey Charles A. Thornton Thurman Wheeler Seng H. Cheng Andrew Leger Bruce M. Wentworth <120> MODULATION OF DYSTROPHIA MYOTONICA-PROTEIN KINASE (DMPK) EXPRESSION <130> BIOL0134WO <140> PCT/US2011/044555 <141> 2011-07-19 <150> 61/365,775 <151> 2010-07-19 <150> 61/365,762 <151> 2010-07-19 <150> 61/478,021 <151> 2011-04-21 <160> 837 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 2877 <212> DNA <213> Homo sapiens <400> 1 aggggggctg gaccaagggg tggggagaag gggaggaggc ctcggccggc cgcagagaga 60 agtggccaga gaggcccagg ggacagccag ggacaggcag acatgcagcc agggctccag 120 ggcctggaca ggggctgcca ggccctgtga caggaggacc ccgagccccc ggcccgggga 180 ggggccatgg tgctgcctgt ccaacatgtc agccgaggtg cggctgaggc ggctccagca 240 gctggtgttg gacccgggct tcctggggct ggagcccctg ctcgaccttc tcctgggcgt 300 ccaccaggag ctgggcgcct ccgaactggc ccaggacaag tacgtggccg acttcttgca 360 gtgggcggag cccatcgtgg tgaggcttaa ggaggtccga ctgcagaggg acgacttcga 420 gattctgaag gtgatcggac gcggggcgtt cagcgaggta gcggtagtga agatgaagca 480 gacgggccag gtgtatgcca tgaagatcat gaacaagtgg gacatgctga agaggggcga 540 ggtgtcgtgc ttccgtgagg agagggacgt gttggtgaat ggggaccggc ggtggatcac 600 gcagctgcac ttcgccttcc aggatgagaa ctacctgtac ctggtcatgg agtattacgt 660 gggcggggac ctgctgacac tgctgagcaa gtttggggag cggattccgg ccgagatggc 720 gcgcttctac ctggcggaga ttgtcatggc catagactcg gtgcaccggc ttggctacgt 780 gcacagggac atcaaacccg acaacatcct gctggaccgc tgtggccaca tccgcctggc 840 cgacttcggc tcttgcctca agctgcgggc agatggaacg gtgcggtcgc tggtggctgt 900 gggcacccca gactacctgt cccccgagat cctgcaggct gtgggcggtg ggcctgggac 960 aggcagctac gggcccgagt gtgactggtg ggcgctgggt gtattcgcct atgaaatgtt 1020 ctatgggcag acgcccttct acgcggattc cacggcggag acctatggca agatcgtcca 1080 ctacaaggag cacctctctc tgccgctggt ggacgaaggg gtccctgagg aggctcgaga 1140 cttcattcag cggttgctgt gtcccccgga gacacggctg ggccggggtg gagcaggcga 1200 cttccggaca catcccttct tctttggcct cgactgggat ggtctccggg acagcgtgcc 1260 cccctttaca ccggatttcg aaggtgccac cgacacatgc aacttcgact tggtggagga 1320 cgggctcact gccatggaga cactgtcgga cattcgggaa ggtgcgccgc taggggtcca 1380 cctgcctttt gtgggctact cctactcctg catggccctc agggacagtg aggtcccagg 1440 ccccacaccc atggaactgg aggccgagca gctgcttgag ccacacgtgc aagcgcccag 1500 cctggagccc tcggtgtccc cacaggatga aacagctgaa gtggcagttc cagcggctgt 1560 ccctgcggca gaggctgagg ccgaggtgac gctgcgggag ctccaggaag ccctggagga 1620 ggaggtgctc acccggcaga gcctgagccg ggagatggag gccatccgca cggacaacca 1680 gaacttcgcc agtcaactac gcgaggcaga ggctcggaac cgggacctag aggcacacgt 1740 ccggcagttg caggagcgga tggagttgct gcaggcagag ggagccacag ctgtcacggg 1800 ggtccccagt ccccgggcca cggatccacc ttcccatcta gatggccccc cggccgtggc 1860 tgtgggccag tgcccgctgg tggggccagg ccccatgcac cgccgccacc tgctgctccc 1920 tgccagggtc cctaggcctg gcctatcgga ggcgctttcc ctgctcctgt tcgccgttgt 1980 tctgtctcgt gccgccgccc tgggctgcat tgggttggtg gcccacgccg gccaactcac 2040 cgcagtctgg cgccgcccag gagccgcccg cgctccctga accctagaac tgtcttcgac 2100 tccggggccc cgttggaaga ctgagtgccc ggggcacggc acagaagccg cgcccaccgc 2160 ctgccagttc acaaccgctc cgagcgtggg tctccgccca gctccagtcc tgtgatccgg 2220 gcccgccccc tagcggccgg ggagggaggg gccgggtccg cggccggcga acggggctcg 2280 aagggtcctt gtagccggga atgctgctgc tgctgctgct gctgctgctg ctgctgctgc 2340 tgctgctgct gctgctgctg ctggggggat cacagaccat ttctttcttt cggccaggct 2400 gaggccctga cgtggatggg caaactgcag gcctgggaag gcagcaagcc gggccgtccg 2460 tgttccatcc tccacgcacc cccacctatc gttggttcgc aaagtgcaaa gctttcttgt 2520 gcatgacgcc ctgctctggg gagcgtctgg cgcgatctct gcctgcttac tcgggaaatt 2580 tgcttttgcc aaacccgctt tttcggggat cccgcgcccc cctcctcact tgcgctgctc 2640 tcggagcccc agccggctcc gcccgcttcg gcggtttgga tatttattga cctcgtcctc 2700 cgactcgctg acaggctaca ggacccccaa caaccccaat ccacgttttg gatgcactga 2760 gaccccgaca ttcctcggta tttattgtct gtccccacct aggaccccca cccccgaccc 2820 tcgcgaataa aaggccctcc atctgcccaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 2877 <210> 2 <211> 14411 <212> DNA <213> Homo sapiens <400> 2 ctcccagccc agcgcctccc accccttttc atagcaggaa aagccggagc ccagggaggg 60 aacggacctg cgagtcacac aactggtgac ccacaccagc ggctggagca ggaccctctt 120 ggggagaaga gcatcctgcc cgcagccagg gcccctcatc aaagtcctcg gtgtttttta 180 aattatcaga actgcccagg accacgtttc ccaggccctg cccagctggg actcctcggt 240 ccttgcctcc tagtttctca ggcctggccc tctcaaggcc caggcacccc aggccggttg 300 gaggccccga cttccactct ggagaaccgt ccaccctgga aagaagagct cagattcctc 360 ttggctctcg gagccgcagg gagtgtgtct tcccgcgcca ccctccaccc cccgaaatgt 420 ttctgtttct aatcccagcc tgggcaggaa tgtggctccc cggccagggg ccaaggagct 480 attttggggt ctcgtttgcc cagggagggc ttggctccac cactttcctc ccccagcctt 540 tgggcagcag gtcacccctg ttcaggctct gagggtgccc cctcctggtc ctgtcctcac 600 caccccttcc ccacctcctg ggaaaaaaaa aaaaaaaaaa aaaaaaagct ggtataaagc 660 agagagcctg agggctaaat ttaactgtcc gagtcggaat ccatctctga gtcacccaag 720 aagctgccct ggcctcccgt ccccttccca ggcctcaacc cctttctccc acccagcccc 780 aacccccagc cctcaccccc tagcccccag ttctggagct tgtcgggagc aagggggtgg 840 ttgctactgg gtcactcagc ctcaattggc cctgtttcag caatgggcag gttcttcttg 900 aaattcatca cacctgtggc ttcctctgtg ctctaccttt ttattggggt gacagtgtga 960 cagctgagat tctccatgca ttccccctac tctagcactg aagggttctg aagggccctg 1020 gaaggaggga gcttgggggg ctggcttgtg aggggttaag gctgggaggc gggagggggg 1080 ctggaccaag gggtggggag aaggggagga ggcctcggcc ggccgcagag agaagtggcc 1140 agagaggccc aggggacagc cagggacagg cagacatgca gccagggctc cagggcctgg 1200 acaggggctg ccaggccctg tgacaggagg accccgagcc cccggcccgg ggaggggcca 1260 tggtgctgcc tgtccaacat gtcagccgag gtgcggctga ggcggctcca gcagctggtg 1320 ttggacccgg gcttcctggg gctggagccc ctgctcgacc ttctcctggg cgtccaccag 1380 gagctgggcg cctccgaact ggcccaggac aagtacgtgg ccgacttctt gcagtggggt 1440 gagtgcctac cctcggggct cctgcagatg gggtgggggt ggggcaggag acaggtctgg 1500 gcacagaggc ctggctgttg ggggggcagg atggcaggat gggcatgggg agatcctccc 1560 atcctggggc tcagagtgtg gacctgggcc ctggggcaac atttctctgt cctatgccac 1620 cactctggag gggcagagta aggtcagcag aggctagggt ggctgtgact cagagccatg 1680 gcttaggagt cacagcaggc taggctgcca acagcctccc atggcctctc tgcaccccgc 1740 ctcagggtca gggtcagggt catgctggga gctccctctc ctaggaccct ccccccaaaa 1800 gtgggctcta tggccctctc ccctggtttc ctgtggcctg gggcaagcca ggagggccag 1860 catggggcag ctgccagggg cgcagccgac aggcaggtgt tcggcgccag cctctccagc 1920 tgccccaaca ggtgcccagg cactgggagg gcggtgactc acgcgggccc tgtgggagaa 1980 ccagctttgc agacaggcgc caccagtgcc ccctcctctg cgatccagga gggacaactt 2040 tgggttcttc tgggtgtgtc tccttctttt gtaggttctg cacccacccc cacccccagc 2100 cccaaagtct cggttcctat gagccgtgtg ggtcagccac cattcccgcc accccgggtc 2160 cctgcgtcct ttagttctcc tggcccaggg cctccaacct tccagctgtc ccacaaaacc 2220 ccttcttgca agggcctcc agggcctggg gccagggctg gaaggaggat gcttccgctt 2280 ctgccagctg ccttgtctgc ccacctcctc cccaagccca ggactcgggc tcactggtca 2340 ctggtttctt tcattcccag caccctgccc ctctggccct catatgtctg gccctcagtg 2400 actggtgttt ggtttttggc ctgtgtgtaa caaactgtgt gtgacacttg tttcctgttt 2460 ctccgccttc ccctgcttcc tcttgtgtcc atctctttct gacccaggcc tggttccttt 2520 ccctcctcct cccatttcac agatgggaag gtggaggcca agaagggcca ggccattcag 2580 cctctggaaa aaccttctcc caacctccca cagcccctaa tgactctcct ggcctccctt 2640 tagtagagga tgaagttggg ttggcagggt aaactgagac cgggtggggt aggggtctgg 2700 cgctcccggg aggagcactc cttttgtggc ccgagctgca tctcgcggcc cctcccctgc 2760 caggcctggg gcgggggagg gggccagggt tcctgctgcc ttaaaagggc tcaatgtctt 2820 ggctctctcc tccctccccc gtcctcagcc ctggctggtt cgtccctgct ggcccactct 2880 cccggaaccc cccggaaccc ctctctttcc tccagaaccc actgtctcct ctccttccct 2940 cccctcccat acccatccct ctctccatcc tgcctccact tcttccaccc ccgggagtcc 3000 aggcctccct gtccccacag tccctgagcc acaagcctcc accccagctg gtcccccacc 3060 caggctgccc agtttaacat tcctagtcat aggaccttga cttctgagag gcctgattgt 3120 catctgtaaa taaggggtag gactaaagca ctcctcctgg aggactgaga gatgggctgg 3180 accggagcac ttgagtctgg gatatgtgac catgctacct ttgtctccct gtcctgttcc 3240 ttcccccagc cccaaatcca gggttttcca aagtgtggtt caagaaccac ctgcatctga 3300 atctagaggt actggataca accccacgtc tgggccgtta cccaggacat tctacatgag 3360 aacgtggggg tggggccctg gctgcacctg aactgtcacc tggagtcagg gtggaaggtg 3420 gaagaactgg gtcttatttc cttctcccct tgttctttag ggtctgtcct tctgcagact 3480 ccgttacccc accctaacca tcctgcacac ccttggagcc ctctgggcca atgccctgtc 3540 ccgcaaaggg cttctcaggc atctcacctc tatgggaggg catttttggc ccccagaacc 3600 ttacacggtg tttatgtggg gaagcccctg ggaagcagac agtcctaggg tgaagctgag 3660 aggcagagag aaggggagac agacagaggg tggggctttc ccccttgtct ccagtgccct 3720 ttctggtgac cctcggttct tttcccccac caccccccca gcggagccca tcgtggtgag 3780 gcttaaggag gtccgactgc agagggacga cttcgagatt ctgaaggtga tcggacgcgg 3840 ggcgttcagc gaggtaagcc gaaccgggcg ggagcctgac ttgactcgtg gtgggcgggg 3900 cataggggtt ggggcggggc cttagaaatt gatgaatgac cgagccttag aacctagggc 3960 tgggctggag gcggggcttg ggaccaatgg gcgtggtgtg gcaggtgggg cggggccacg 4020 gctgggtgca gaagcgggtg gagttgggtc tgggcgagcc cttttgtttt cccgccgtct 4080 ccactctgtc tcactatctc gacctcaggt agcggtagtg aagatgaagc agacgggcca 4140 ggtgtatgcc atgaagatca tgaacaagtg ggacatgctg aagaggggcg aggtgagggg 4200 ctgggcggac gtggggggct ttgaggatcc gcgccccgtc tccggctgca gctcctccgg 4260 gtgccctgca ggtgtcgtgc ttccgtgagg agagggacgt gttggtgaat ggggaccggc 4320 ggtggatcac gcagctgcac ttcgccttcc aggatgagaa ctacctggtg agctccgggc 4380 cggggtgact aggaagaggg acaagagccc gtgctgtcac tggacgagga ggtggggaga 4440 ggaagctcta ggattggggg tgctgcccgg aaacgtctgt gggaaagtct gtgtgcggta 4500 agagggtgtg tcaggtggat gaggggcctt ccctatctga gacggggatg gtgtccttca 4560 ctgcccgttt ctggggtgat ctgggggact cttataaaga tgtctctgtt gcggggggtc 4620 tcttacctgg aatgggatag gtcttcagga attctaacgg ggccactgcc tagggaagga 4680 gtgtctggga cctattctct gggtgttggg tggcctctgg gttctctttc ccagaacatc 4740 tcagggggag tgaatctgcc cagtgacatc ccaggaaagt ttttttgttt gtgttttttt 4800 ttgaggggcg ggggcggggg ccgcaggtgg tctctgattt ggcccggcag atctctatgg 4860 ttatctctgg gctggggctg caggtctctg cccaaggatg gggtgtctct gggaggggtt 4920 gtcccagcca tccgtgatgg atcagggcct caggggacta ccaaccaccc atgacgaacc 4980 ccttctcagt acctggtcat ggagtattac gtgggcgggg acctgctgac actgctgagc 5040 aagtttgggg agcggattcc ggccgagatg gcgcgcttct acctggcgga gattgtcatg 5100 gccatagact cggtgcaccg gcttggctac gtgcacaggt gggtgcagca tggccgaggg 5160 gatagcaagc ttgttccctg gccgggttct tggaaggtca gagcccagag aggccagggc 5220 ctggagaggg accttcttgg ttggggccca ccggggggtg cctgggagta ggggtcagaa 5280 ctgtagaagc cctacagggg cggaacccga ggaagtgggg tcccaggtgg cactgcccgg 5340 aggggcggag cctggtggga ccacagaagg gaggttcatt tatcccaccc ttctcttttc 5400 ctccgtgcag ggacatcaaa cccgacaaca tcctgctgga ccgctgtggc cacatccgcc 5460 tggccgactt cggctcttgc ctcaagctgc gggcagatgg aacggtgagc cagtgccctg 5520 gccacagagc aactggggct gctgatgagg gatggaaggc acagagtgtg ggagcgggac 5580 tggatttgga ggggaaaaga ggtggtgtga cccaggctta agtgtgcatc tgtgtggcgg 5640 agtattagac caggcagagg gaggggctaa gcatttgggg agtggttgga aggagggccc 5700 agagctggtg ggcccagagg ggtgggccca agcctcgctc tgctcctttt ggtccaggtg 5760 cggtcgctgg tggctgtggg caccccagac tacctgtccc ccgagatcct gcaggctgtg 5820 ggcggtgggc ctgggacagg cagctacggg cccgagtgtg actggtgggc gctgggtgta 5880 ttcgcctatg aaatgttcta tgggcagacg cccttctacg cggattccac ggcggagacc 5940 tatggcaaga tcgtccacta caaggtgagc acggccgcag ggagacctgg cctctcccgg 6000 taggcgctcc caggctatcg cctcctctcc ctctgagcag gagcacctct ctctgccgct 6060 ggtggacgaa ggggtccctg aggaggctcg agacttcatt cagcggttgc tgtgtccccc 6120 ggagacacgg ctgggccggg gtggagcagg cgacttccgg acacatccct tcttctttgg 6180 cctcgactgg gatggtctcc gggacagcgt gccccccttt acaccggatt tcgaaggtgc 6240 caccgacaca tgcaacttcg acttggtgga ggacgggctc actgccatgg tgagcggggg 6300 cggggtaggt acctgtggcc cctgctcggc tgcgggaacc tccccatgct ccctccataa 6360 agttggagta aggacagtgc ctaccttctg gggtcctgaa tcactcattc cccagagcac 6420 ctgctctgtg cccatctact actgaggacc cagcagtgac ctagacttac agtccagtgg 6480 gggaacacag agcagtcttc agacagtaag gccccagagt gatcagggct gagacaatgg 6540 agtgcagggg gtgggggact cctgactcag caaggaaggt cctggagggc tttctggagt 6600 ggggagctat ctgagctgag acttggaggg atgagaagca ggagaggact cctcctccct 6660 taggccgtct ctcttcaccg tgtaacaagc tgtcatggca tgcttgctcg gctctgggtg 6720 cccttttgct gaacaatact ggggatccag cacggaccag atgagctctg gtccctgccc 6780 tcatccagtt gcagtctaga gaattagaga attatggaga gtgtggcagg tgccctgaag 6840 ggaagcaaca ggatacaaga aaaaatgatg gggccaggca cggtggctca cgcctgtaac 6900 cccagcaatt tggcaggccg aagtgggtgg attgcttgag cccaggagtt cgagaccagc 6960 ctgggcaatg tggtgagacc cccgtctcta caaaaatgtt ttaaaaattg gttgggcgtg 7020 gtggcgcatg cctgtatact cagctactag ggtggccgac gtgggcttga gcccaggagg 7080 tcaaggctgc agtgagctgt gattgtgcca ctgcactcca gcctgggcaa cggagagaga 7140 ctctgtctca aaaataagat aaactgaaat taaaaaatag gctgggctgg ccgggcgtgg 7200 tggctcacgc ctgtaatctc agcactttgg gaggccgagg cgggtggatc acgaggtcag 7260 gagatcgaga ccatcttggc taacacggtg aaaccccatc tctcctaaaa atacaaaaaa 7320 ttagccaggc gtggtggcgg gcgcctgtag tcccagctac tcaggaggct gaggcaggag 7380 aatggcgtga acccgggagg cagagtttgc agtgagccga gatcgtgcca ctgcactcca 7440 gcctgggcga cagagcgaga ctctgtctca gaaaaaaaaa aaaaaaaaaa aaaaaatagg 7500 ctggaccgcg gccgggcgct gtggctcatg cctgtaatcc cagcactttg ggagtccaag 7560 gccggtgggt catgagatca ggagttttga gactaggctg gccaacacgg tgaaaccccg 7620 tctctactaa aaatacaaga aaattagctg ggtgtggtct cgggtgcctg taattccagt 7680 tactggggaa gctgaggcag gagaattgct tgaacctggg aggcagagtt tgcagtgagc 7740 caagatcatg ccactacact ccagtctggg tgacagagtg agactctgtc tcaaaaaaaa 7800 aaaaaaaaaa aagggttggg caaggtggtt cacgcctgta atcccagaac tttgggaggc 7860 tgaggcaggc agatcactgg aagtcaggag ttcaagacca gcctggccaa catggtgaaa 7920 ccctgtgtct actaaaaata caaaatttag ccaggcttgg tggcgtatgc ctgtaatgcc 7980 agctactcag gaggctgagg caggagaatc gcttgattga acctgggagg cagagtttgc 8040 agtgggctgg ggttgtgcca ctgcactcta ggctgggaga cagcaagact ccatctaaaa 8100 aaaaaaaaca gaactgggct gggcacagtg gcttatattt gtaatcccag cactttggga 8160 ggctgaggtt ggaggactgc ttgagcccag agtttgggac tacaacagct gaggtaggcg 8220 gatcacttga ggtcagaaga tggagaccag cctggccagc gtggcgaaac cccgtctcta 8280 ccaaaaatat aaaaaattag ccaggcgtgg tagagggcgc ctgtaatctc agctactcag 8340 gacgctgagg caggagaatc gcctgaacct gggaggcgga ggttgcagtg agctgagatt 8400 gcaccactgc actccagcct gggtaacaga gcgagactcc gtatcaaaga aaaagaaaaa 8460 agaaaaaatg ctggaggggc cactttagat aagccctgag ttggggctgg tttgggggga 8520 acatgtaagc caagatcaaa aagcagtgag gggcccgccc tgacgactgc tgctcacatc 8580 tgtgtgtctt gcgcaggaga cactgtcgga cattcgggaa ggtgcgccgc taggggtcca 8640 cctgcctttt gtgggctact cctactcctg catggccctc aggtaagcac tgccctggac 8700 ggcctccagg ggccacgagg ctgcttgagc ttcctgggtc ctgctccttg gcagccaatg 8760 gagttgcagg atcagtcttg gaaccttact gttttgggcc caaagactcc taagaggcca 8820 gagttggagg accttaaatt ttcagatcta tgtacttcaa aatgttagat tgaattttaa 8880 aacctcagag tcacagactg ggcttcccag aatcttgtaa ccattaactt ttacgtctgt 8940 agtacacaga gccacaggac ttcagaactt ggaaaatatg aagtttagac ttttacaatc 9000 agttgtaaaa gaatgcaaat tctttgaatc agccatataa caataaggcc atttaaaagt 9060 attaatttag gcgggccgcg gtggctcacg cctgtaatcc tagcactttg ggaggccaag 9120 gcaggtggat catgaggtca ggagatcgag accatcctgg ctaacacggt gaaaccccgt 9180 ctctactaaa aatacaaaaa aattagccgg gcatggtggc gggcgcttgc ggtcccagct 9240 acttgggagg cgaggcagga gaatggcatg aacccgggag gcggagcttg cagtgagccg 9300 agatcatgcc actgcactcc agcctgggcg acagagcaag actccgtctc aaaaaaaaaa 9360 aaaaaaaagt atttatttag gccgggtgtg gtggctcacg cctgtaattc cagtgctttg 9420 ggaggatgag gtgggtggat cacctgaggt caggagttcg agaccagcct gaccaacgtg 9480 gagaaacctc atctctacta aaaaacaaaa ttagccaggc gtggtggcat atacctgtaa 9540 tcccagctac tcaggaggct gaggcaggag aatcagaacc caggaggggg aggttgtggt 9600 gagctgagat cgtgccattg cattccagcc tgggcaacaa gagtgaaact tcatctcaaa 9660 aaaaaaaaaa aaaaagtact aatttacagg ctgggcatgg tggctcacgc ttggaatccc 9720 agcactttgg gaggctgaag tggacggatt gcttcagccc aggagttcaa gaccagcctg 9780 agcaacataa tgagaccctg tctctacaaa aaattgaaaa aatcgtgcca ggcatggtgg 9840 tctgtgcctg cagtcctagc tactcaggag tctgaagtag gagaatcact tgagcctgga 9900 gtttgaggct tcagtgagcc atgatagatt ccagcctagg caacaaagtg agacctggtc 9960 tcaacaaaag tattaattac acaaataatg cattgcttat cacaagtaaa ttagaaaata 10020 cagataagga aaaggaagtt gatatctcgt gagctcacca gatggcagtg gtccctggct 10080 cacacgtgta ctgacacatg tttaaatagt ggagaacagg tgtttttttg gtttgttttt 10140 ttccccttcc tcatgctact ttgtctaaga gaacagttgg ttttctagtc agcttttatt 10200 actggacaac attacacata ctatacctta tcattaatga actccagctt gattctgaac 10260 cgctgcgggg cctgaacggt gggtcaggat tgaacccatc ctctattaga acccaggcgc 10320 atgtccagga tagctaggtc ctgagccgtg ttcccacagg agggactgct gggttggagg 10380 ggacagccac ttcatacccc agggaggagc tgtccccttc ccacagctga gtggggtgtg 10440 ctgacctcaa gttgccatct tggggtccca tgcccagtct taggaccaca tctgtggagg 10500 tggccagagc caagcagtct ccccatcagg tcggcctccc tgtcctgagg ccctgagaag 10560 aggggtctgc agcggtcaca tgtcaaggga ggagatgagc tgaccctaga acatgggggt 10620 ctggacccca agtccctgca gaaggtttag aaagagcagc tcccaggggc ccaaggccag 10680 gagaggggca gggcttttcc taagcagagg aggggctatt ggcctacctg ggactctgtt 10740 ctcttcgctc tgctgctccc cttcctcaaa tcaggaggtc ttggaagcag ctgcccctac 10800 ccacaggcca gaagttctgg ttctccacca gagaatcagc attctgtctc cctccccact 10860 ccctcctcct ctccccaggg acagtgaggt cccaggcccc acacccatgg aactggaggc 10920 cgagcagctg cttgagccac acgtgcaagc gcccagcctg gagccctcgg tgtccccaca 10980 ggatgaaaca gtaagttggt ggaggggagg gggtccgtca gggacaattg ggagagaaaa 11040 ggtgagggct tcccgggtgg cgtgcactgt agagccctct agggacttcc tgaacagaag 11100 cagacagaaa ccacggagag acgaggttac ttcagacatg ggacggtctc tgtagttaca 11160 gtggggcatt aagtaagggt gtgtgtgttg ctggggatct gagaagtcga tctttgagct 11220 gagcgctggt gaaggagaaa caagccatgg aaggaaaggt gccaagtggt caggcgagag 11280 cctccagggc aaaggccttg ggcaggtggg aatcctgatt tgttcctgaa aggtagtttg 11340 gctgaatcat tcctgagaag gctggagagg ccagcaggaa acaaaaccca gcaaggcctt 11400 ttgtcgtgag ggcattaggg agctggaggg attttgagca gcagagggac ataggttgtg 11460 ttagtgtttg agcaccagcc ctctggtccc tgtgtagatt tagaggacca gactcaggga 11520 tggggctgag ggaggtaggg aagggagggg gcttggatca ttgcaggagc tatggggatt 11580 ccagaaatgt tgaggggacg gaggagtagg ggataaacaa ggattcctag cctggaacca 11640 gtgcccaagt cctgagtctt ccaggagcca caggcagcct taagcctggt ccccatacac 11700 aggctgaagt ggcagttcca gcggctgtcc ctgcggcaga ggctgaggcc gaggtgacgc 11760 tgcgggagct ccaggaagcc ctggaggagg aggtgctcac ccggcagagc ctgagccggg 11820 agatggaggc catccgcacg gacaaccaga acttcgccag gtcgggatcg gggccggggc 11880 cggggccggg atgcgggccg gtggcaaccc ttggcatccc ctctcgtccg gcccggacgg 11940 actcaccgtc cttacctccc cacagtcaac tacgcgaggc agaggctcgg aaccgggacc 12000 tagaggcaca cgtccggcag ttgcaggagc ggatggagtt gctgcaggca gagggagcca 12060 caggtgagtc cctcatgtgt ccccttcccc ggaggaccgg gaggaggtgg gccgtctgct 12120 ccgcggggcg tgtatagaca cctggaggag ggaagggacc cacgctgggg cacgccgcgc 12180 caccgccctc cttcgcccct ccacgcgccc tatgcctctt tcttctcctt ccagctgtca 12240 cgggggtccc cagtccccgg gccacggatc caccttccca tgtaagaccc ctctctttcc 12300 cctgcctcag acctgctgcc cattctgcag atcccctccc tggctcctgg tctccccgtc 12360 cagatatagg gctcacccta cgtctttgcg actttagagg gcagaagccc tttattcagc 12420 cccagatctc cctccgttca ggcctcacca gattccctcc gggatctccc tagataacct 12480 ccccaacctc gattcccctc gctgtctctc gccccaccgc tgagggctgg gctgggctcc 12540 gatcgggtca cctgtccctt ctctctccag ctagatggcc ccccggccgt ggctgtgggc 12600 cagtgcccgc tggtggggcc aggccccatg caccgccgcc acctgctgct ccctgccagg 12660 gtacgtccgg ctgcccacgc ccccctccgc cgtcgcgccc cgcgctccac ccgccccttg 12720 ccacccgctt agctgcgcat ttgcggggct gggcccacgg caggagggcg gatcttcggg 12780 cagccaatca acacaggccg ctaggaagca gccaatgacg agttcggacg ggattcgagg 12840 cgtgcgagtg gactaacaac agctgtaggc tgttggggcg ggggcggggc gcagggaaga 12900 gtgcgggccc acctatgggc gtaggcgggg cgagtcccag gagccaatca gaggcccatg 12960 ccgggtgttg acctcgccct ctccccgcag gtccctaggc ctggcctatc ggaggcgctt 13020 tccctgctcc tgttcgccgt tgttctgtct cgtgccgccg ccctgggctg cattgggttg 13080 gtggcccacg ccggccaact caccgcagtc tggcgccgcc caggagccgc ccgcgctccc 13140 tgaaccctag aactgtcttc gactccgggg ccccgttgga agactgagtg cccggggcac 13200 ggcacagaag ccgcgcccac cgcctgccag ttcacaaccg ctccgagcgt gggtctccgc 13260 ccagctccag tcctgtgatc cgggcccgcc ccctagcggc cggggaggga ggggccgggt 13320 ccgcggccgg cgaacggggc tcgaagggtc cttgtagccg ggaatgctgc tgctgctgct 13380 gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctggggg gatcacagac 13440 catttctttc tttcggccag gctgaggccc tgacgtggat gggcaaactg caggcctggg 13500 aaggcagcaa gccgggccgt ccgtgttcca tcctccacgc acccccacct atcgttggtt 13560 cgcaaagtgc aaagctttct tgtgcatgac gccctgctct ggggagcgtc tggcgcgatc 13620 tctgcctgct tactcgggaa atttgctttt gccaaacccg ctttttcggg gatcccgcgc 13680 ccccctcctc acttgcgctg ctctcggagc cccagccggc tccgcccgct tcggcggttt 13740 ggatatttat tgacctcgtc ctccgactcg ctgacaggct acaggacccc caacaacccc 13800 aatccacgtt ttggatgcac tgagaccccg acattcctcg gtatttattg tctgtcccca 13860 cctaggaccc ccacccccga ccctcgcgaa taaaaggccc tccatctgcc caaagctctg 13920 gactccacag tgtccgcggt ttgcgttgtg ggccggaggc tccgcagcgg gccaatccgg 13980 aggcgtgtgg aggcggccga aggtctggga ggagctagcg ggatgcgaag cggccgaatc 14040 agggttgggg gaggaaaagc cacggggcgg ggctttggcg tccggccaat aggagggcga 14 100 gcgggccacc cggaggcacc gcccccgccc agctgtggcc cagctgtgcc accgagcgtc 14160 gagaagaggg ggctgggctg gcagcgcgcg cggccatcct ccttccactg cgcctgcgca 14220 cgccacgcgc atccgctcct gggacgcaag ctcgagaaaa gttgctgcaa actttctagc 14280 ccgttccccg cccctcctcc cggccagacc cgccccccct gcggagccgg gaattccgag 14340 gggcggagcg caggccgaga tggggaatgt gggggcctgc agaggaccct ggagacggag 14400 gcgtgcagaa g 14411 <210> 3 <211> 15000 <212> DNA <213> Mus musculus <400> 3 cagtgtcccc actgcccaag gctggctcca tcacgtaccg ctttggctca gctggccagg 60 acacacagtt ctgcctgtgg gacctcacag aagatgtgct ctcccctcat ccgtctctgg 120 cccgtacccg cacccttccg ggcacacctg gtgccacccc accagcttct ggtagttctc 180 gggccggaga gacaggtgca ggccccctgc cccgctccct gtctcgttcc aacagtctcc 240 cacacccagc tggtggtggc aaggctggtg ggcctagtgc atcgatggag cctggcatac 300 cattcagcat tggccgcttt gccacactga ccctgcagga gcggcgggac cggggagctg 360 agaaggaaca caaacgctac catagcctgg gaaacatcag ccgcggtggc agtgggggca 420 atagcagcaa tgacaagctc agtggtcctg ccccccgaag ccgattggac ccagctaagg 480 tgctgggcac ggcactgtgc cctcggatcc atgaggtgcc actgctggag cctctcgtgt 540 gcaagaagat tgctcaggaa cgcctgaccg tgctactgtt cctggaggat tgtatcatca 600 ctgcctgcca agagggcctc atctgcacct gggcccggcc aggcaaggcg gtgagtccgc 660 acctgcccaa gcgctgaggg gcaccagttc tgtccctacc ggatgccagt tatccgtcag 720 cagaaaggtc aggtatagga gacagaatgg ggggaaccac agctaacgtc tttagagcct 780 ctgctggccc atatggctca tccttagtac ttcacactca aggcagaacc tgtgtttata 840 ggaaatctga agtgtagatg gtgaaacttt attcaggtct agggatgtga ttgagctggg 900 ggcccacttc tggcctgcct cttagacact gtttctgagc cagctgctga aggcctggat 960 gggaattagc cagggtccag gcctgcactt cctcttgctg ctgtgtggtc ctggtcattg 1020 ggtctcacag atgggctgtg cagtggctgt gctcttagtt ggtgaggtgc aggcctgtca 1080 cctggtcagg cttgagcatg tggtctcagt gtctaggacc ctactctgcc ctcagtcctt 1140 cagtcccttg ctttggaagg ctagagtcca gaagccttag aacgtcaggc agttgcagag 1200 ccactgccag gctagtaggg ctgcgggagt tgactgagtt ctcacagaca cccctctgtc 1260 tccctagttc acagacgagg agaccgaggc ccaggcaggg caagcaagtt ggcccaggtc 1320 acccagcaag tcagttgtag aggtaggaca acccctgaag ctgcaagtgg accccagttt 1380 cttttctctc cactgtcgtc ccctgtatgc ccaggacacc tggggccaca ttactgtgga 1440 agtgctactc tgggtcagtg gagacggccg agctgtttgt tcctagctag gacagcagct 1500 ttaggcctgg ggggcagatc ccagctgggg cagcagctcc aaggcctttg ggtggctcct 1560 tctccgggtt ctggcagaag cccaggtgct gtctaatcca cctttctcct cttgttctcc 1620 ccagggcatc tcctcccaac caggcagctc ccccagtggc actgtggtgt gaaatgtgga 1680 tgtcccatgt tcccggcctc ctagccataa ccctccccgc tgacctcaag aatcactgta 1740 ttaacaagac taatcatgat ggaaggactg ctccaagccc cacgctgcac acatactggg 1800 ggtcccctag gttggcccag ccatggggat gtagtgtcct gtgtggcctt ggccctgtcc 1860 tccacccact gccaagtaca atgacctgtt ctctgaaaca tcagtgttaa ccatatccct 1920 gtcccagcat gtgactgttc actcctggga gagacttagc ccacagtacc cctgggtgag 1980 agggcagggc aggggccatc cccactcctg cccaaactcc accccttgct atggtctgtg 2040 attttgaaag tgttaaatta tggaagccct gagggccctc cttgttcccc tggacctctt 2100 atttatacta aagtccttgt ttgcacagtg tttctgttcc ctggggcagg gtagggtggg 2160 ggttgcagta cttggcctcc aagctgtgct ctgaccaaag gaagcccaat cttagctgtt 2220 tccccatccc tagccccgag cagagagccc tctgaaagat gagtctcgac ccccaaagtc 2280 aagaggctga gatggccttc ctactaggtc cttggagatg tttgaaactt gttttaaaca 2340 ccaggactat ccaagcatgc tctccttggg gagaggagga tgctggaatt gactgcactc 2400 cctgcctcct ctgaacatgc ctttgcagtc tgctgcccct ggcccattta tgactggcca 2460 tctagtgcca gctggaggtc atgatttcct ccccagagaa ctggccaccc tagaaagaag 2520 ctaacttgtc gcctggcttg ctgtccaggc agctccgccc tcaaccccta aaatgtttct 2580 gtctctaatc ctagcccagg caggaatgtg gctgccccgg cctgtggcca aggagctatt 2640 ttggggttct cttttgctta aggagggcct ggatccacca cttgcctccc ccaggctggg 2700 gccagcaggt cacccctggc cctggcggct gagcaaactc tctcctgatc ttccttctac 2760 ctcctgccaa aaaatggggg ggcgggtaat acagcaggca caggggctaa atttaactgt 2820 cccaaagtcg gaatccattg ctgagtcacg aagaagctgc ccctggcctt tgcccccccc 2880 actaccccct caccccctgt tgcccaggca tcagcccttt cccccaaccc ctcccagctc 2940 tgagtctata gactggctct cctgggcact gacacctccc acctgtaact ccctgtgctc 3000 tctttatggg tgggtagagt caatgggggg gggcaaccct ggagtattac tctgtcccct 3060 gacattgggc tctgaagagt tttgaggggc cctggaagaa gggagttggg gtgttggctc 3120 aggaggggtt aaaaactggg aggcgggagg ggggctgggc caaggggtgg agaaaagagg 3180 aggaggcctt aagcatagaa ctggccagag agacccaagg gatagtcagg gacgggcaga 3240 catgcagcta gggttctggg gcctggacag gggcagccag gccctgtgac gggaagaccc 3300 cgagctccgg cccggggagg ggccatggtg ttgcctgccc aacatgtcag ccgaagtgcg 3360 gctgaggcag ctccagcagc tggtgctgga cccaggcttc ctgggactgg agcccctgct 3420 cgaccttctc ctgggcgtcc accaggagct gggtgcctct cacctagccc aggacaagta 3480 tgtggccgac ttcttgcagt ggggtgagta tggataggaa gcctggggtt gggtgcaagg 3540 cagaggtggg tctacagggc aagaatgggc tatggagggg caggagggcc tggaaagggc 3600 tttttgtaag ggagccaagc agagctcatg acctgacccc aagctcccct ggtgaggcac 3660 cagggtcagt gaggccacct atgactcagc cagtgcaggc tggggtgggc atagcctcct 3720 gctatctcag cacccacact aggacctggc agctttctct tttaggaccc ttggctcctc 3780 aaactggctt catagccctc cccagtttcc cagagtgtgg ggagggacag cgtggggcag 3840 ctgccagggt gtggcccata ggcaggtgtt tggcgtctgc ctccccagct gccctgacag 3900 gtgtccagga gctatgaggg cactgtgact cacagaggcc ctgggggaga accagcccgg 3960 cagacaggcg ccaccgagca ccctttctgt tccccaaatt aagaggaagg aacaacttca 4020 gcttctgagt gtgcccatcc ctagcactct gatcccgccc agcctttgtg ggccagattg 4080 gtcatccctc ctggcttctc atctgctttt gtggttctag ctcaagacct ctaattcctc 4140 tgctgactta aatgcccttc cccagaggtc ttctcaggcc tagtggacaa gcttggagcc 4200 ttatctgctc ctgcccaaca ttgagccaaa gctccagctt accccagctt ccttacaagt 4260 aacgacctgt tttgttgctc tgtgcctatt attaagggtc caggtcttga ttcttggctg 4320 tctgcccatg tgtgtgaccc tagtgcattc tcccctcctc ccccgtttca cagatggaaa 4380 ggttgaggcc atcggttaga ctgctaagcc tgtgaaagac tttttctcct ctccagtctt 4440 tagtgtctcc ctcaaccttt cttttgaagg atggggtttg ggctggcagg gtaaactgag 4500 aactggggtg ggggcagggg gtctgaccct ctgggaagga gcagtccttt tgtggcctga 4560 gcagcatcct gtgggcccct cccctgccag gcctgggcgg gggagggggc ctgggttccc 4620 gctgccttaa aagggctcaa cgccttggct ctctcctcct ccccaccccc cagccttggc 4680 cctagctgta tcttccccgg ctgcccactt tcccaaaccc ctttcttctc tgtgacccca 4740 tctccccgct tccccacacg tccctcctcc atccttactc cccggcctta gaacttccct 4800 aagggagatc tgacctccct ctgcccaccc cgcaccccca gtcgccagcc tcagacctag 4860 ctgctctccc ctctggctga accaccctag cacaggacct tataccctgg agctttggtt 4920 ataagaagac tctccttcac cctttggaaa ccaagaaagc ccttccaaca gtgtccagga 4980 tgctggaggg cagtgaccct cccccacttc ttcttcgtgc tggctgtgct gacacagctc 5040 cagttcgagg ttgtggcccg agacattaag tgagagcccc gggtgacctg acttagcacc 5100 ctgatcatca catgggagtg aaaggcctga tgcgccagct tctcccactg cctccctttc 5160 tgccctgcaa ccctgtggaa acaggcagtt ctgggtccca caaacatcac agaggttttg 5220 aaagcagaat cctaaagccg atttaagggg cagaaggaag gaggctataa agtcactacc 5280 cttaccgcta gtgttctgat gacccttggt tcttcttccc ccacccccgc ccagtggagc 5340 ccattgcagc aaggcttaag gaggtccgac tgcagaggga tgattttgag attttgaagg 5400 tgatcgggcg tggggcgttc agcgaggtga gtcttcagtg gcctgggaat ggaactttac 5460 ttgatgtggg tggggcataa cagctggggc agagccttaa aaattgatga atgagcttga 5520 atttaaggct ggaggggtgg gggcggagct tgtggtcagt gggcggtgtg cacgtgaggg 5580 cggggctaag gttgggtgga gataagggtg gagtcctgtc tgggtgagcc ttgctggttt 5640 tccctgccac ctcttgctgt catctcggtt ccgtatttag gtagcggtgg tgaagatgaa 5700 acagacgggc caagtgtatg ccatgaagat tatgaataag tgggacatgc tgaagagagg 5760 cgaggtgagg gccagggatt agggcagcgc cctcatctct ccaactcacc tcctgtagct 5820 tctctcctac ctcacaggtg tcgtgcttcc gggaagaaag ggatgtatta gtgaaagggg 5880 accggcgctg gatcacacag ctgcactttg ccttccagga tgagaactac ctggtaagct 5940 ccgggttcag gtgactagga aagagtgaca gttacatcgc cccaagtcaa gaaggctgga 6000 gaagggagaa gctgctgtag atcggggggg tgggggtggg ggggacacac acaggggatg 6060 ggggacgggg gtaggattgt gtctcaagta taggagagac cttccttgag acaggagtga 6120 tatctggttt ggcctttgga tggggcgctc tctcactgtg cgggggtcct ctgtgcttgg 6180 gaacggggtg tctttgggag tcttgggggc taccaaaccc ctgtgacaca cccgctccca 6240 gtacctggtc atggaatact acgtgggcgg ggacctgcta acgctgctga gcaagtttgg 6300 ggagcggatc cccgccgaga tggctcgctt ctacctggcc gagattgtca tggccataga 6360 ctccgtgcac cggctgggct acgtgcacag gtgggcgtgg cggggccctt ggagggttag 6420 cagaatttgt gtgggaagga agggtacctg aaggtcagat cccattgggg acagaatcgg 6480 ggtctagaat tgtagaatcc tgggtggggt ggaagtggat cgagctgacg ggccctaaga 6540 gggaaggttt tcaagaaagc acactttccc tcttctctct atgcacaggg acatcaaacc 6600 agataacatt ctgctggacc gatgtgggca cattcgcctg gcagacttcg gctcctgcct 6660 caaactgcag cctgatggaa tggtaagaag agcctggcga aactctcctc attggtgaag 6720 gaccggatta gggggcgggg ctgggttgag gagcaggagg ggagcttggt ctgggatgtc 6780 ctgcgcacca tatttggaca gtcaagggaa aggttttaag cattcaggtc tgattggcac 6840 aggtgaggtc gctggtggct gtgggcaccc cggactacct gtctcctgag attctgcagg 6900 ccgttggtgg agggcctggg gcaggcagct acgggccaga gtgtgactgg tgggcactgg 6960 gcgtgttcgc ctatgagatg ttctatgggc agaccccctt ctacgcggac tccacagccg 7020 agacatatgc caagattgtg cactacaggg tgagcacaag caccatgcag gggggctgac 7080 ttagtggctt gtgctcccag actgtctttt ttaaaagata tttatttata tgtgtgtgtt 7140 ttctgtgtat gtatatctgt gcactgagta ggtgtgcgaa ggtcagaggg catgggatcc 7200 cctggaactg gagtcacaga ctattgtgtg ctgccatgct gagtgctggg aaacagaacc 7260 ttgatcactc tgcaagagca gccagtgcac tgaaacgaca gagccagctc tgcagcccag 7320 ggctaactgt tgcttttctt tctaaatagg aacacttgtc gctgccgctg gcagacacag 7380 ttgtccccga ggaagctcag gacctcattc gtgggctgct gtgtcctgct gagataaggc 7440 taggtcgagg tggggcaggt gatttccaga aacatccttt cttctttggc cttgattggg 7500 agggtctccg agacagtgta ccccccttta caccagactt cgagggtgcc acggacacat 7560 gcaatttcga tgtggtggag gaccggctca ctgccatggt gagcgggggc ggggtacgta 7620 cctgcagttc ctgatccgtt gaggggactt ccctagcctc ttccataaaa ttggggtgat 7680 tggccaggtg tggtggtgca tacctttaat cgtagaactt cataggcaga ggcaggtggc 7740 tctctggtaa atcaaggcca tcttggtcta catagtgact tctaggccag tcaggagtga 7800 gatcctccct tgaaaaataa aaaagggggt gttgaccttc ctgggtccca aattattatc 7860 ctagagcact gctatgtatc cactcaggta tgaggacaca caggtgacca gtcccaaaga 7920 cagtgagtga ggcctcactc ttggcagtac taaaattgat tgtagggggc tgggctcttg 7980 acccagcctg gaaagtgctg gagggcttcc tggaggagga gactagctga gcccagaagg 8040 atgcaggaga tcctttctcg ggtgagtgct ctcagcattt taacaagctc taggccctgc 8100 agagagaagt ctggtgtggg cagagcccca atagaaagca acaagataga agagaaaatg 8160 gtggagtttg ttagtggggg cagttatgcc gtgaacatag aggggcgaag ggccatctcg 8220 gataactgct agccacaaga gccctgtctg tcttcctagg agacgctgtc agacatgcag 8280 gaagacatgc cccttggggt gcgcctgccc ttcgtgggct actcctactg ctgcatggcc 8340 ttcaggtgag cacgactgcc ccctgctggg gcctgtgtgc aggcccacca cagccactca 8400 attgaaggct cagtcttcaa accaagtatt cctaggagct gtctaagtta ggctttctgc 8460 tgctgcgatg aaccctgact aaaagcaagc tggggaggaa aaggcttatc gggcttacgt 8520 ttccacatgg gagcccatca ctgaaggaag ccaggacagg aactcacagc ggggcaggaa 8580 cgtggagctg atgcagaggc aatggagggg agctgcttac tgacttgatc cttatgtctt 8640 cctcagcctg tttccttgta gagcccagga ccaccaggcc agtgagggct ccactcacaa 8700 tgggctgagc tctcatctat gatcactagt tatgaaaatg cccgataggc ttgcctgcag 8760 cttcagtttt tgaggcactt tccttccttc cttccttcct tccttccttc cttccttcct 8820 ttctttcttt ctttctttct ttctttcttt ctttctttct ttctttcttt ctttctttct 8880 tagtctttta gagacagggt ctttctatgt agctctggct gtcttggaat tcattctgta 8940 gaccaggctg gtcttattta tttattttat gtatgtgagt ccactatcac tgtcctcaga 9000 cacaccagaa gagggcatca gatcccatta cagatggctg tgagccacca tgtggttgct 9060 gggaattgaa ctcaggacct ctggaagagc agccagtgct cctgccctgt agaggcattt 9120 tcttcatgaa ggctgtctcc tctctgatga cttgatgact ctagcttgtt gtgtcaagtg 9180 gacataagac taggaaagca gctacacatg cactttgttt atttttgttt tgctttttga 9240 gactgggtct ctccatctca tagctctggc catcctgcct ggtgacattc cagtccagtt 9300 gtataaccta agaatctgag actcagcctt gcagaatcct gctattaacg ggtctaggac 9360 actccataga atccaggatc ttagaaaaac aaacctgaag tgtgacagtt tattttaaga 9420 acacaattgg agcacataac aataatacaa cttttcagtt ttaaaaagtt ttctgtcttg 9480 ttttttgagg caggagctcc ttaatatagt ctaagccgcc ctgcgagtgc tgtgattgat 9540 gggcatgtac caccatgcct agtcaataaa gcctttaaaa agcatccgtt atgctggctg 9600 tggtgccaca aacctgtaat cccagcactt agaaggtaga ggcaagatta tcagaaattc 9660 aaggccatcc tgggctatac agtaatctaa ggctagcctg gtctacaaga gactctgtct 9720 aaagaaacaa aagataaata gcacccacta ttgctaggca atataaccct ataaccccac 9780 cattgaggag gctgaggctg gagcatcact gcaaatttga ggccaggatg gtcaacaaat 9840 aagtcccaga gctggcatag aggaactctg tctcaacaat aaagagaact tatctagcat 9900 ttatgagggt aaataaaaat ttaccattgc cacaaaaaat gtaaatgaag agactgcttt 9960 taggagtgaa ctgggaagca gggaacactt agaggatgct cactcacaca ggtatccacc 10020 atcaggcatg cctcaggcct gcacagggaa ggacaacttg tttcatgatt tgcaagcagc 10080 atcccatgct ccttagagcg ggttgggccc agcccaccct ctgtggagtt atcgctcagc 10140 caggcagcaa ggcagccaag gtgctgaggc cctggcagtc tgctctcttc tctgctctga 10200 acctccttta gctttagcct aggagcctgg cctggtgccc acaggctagg gagtccctag 10260 cctcttcctc ttctcagaga caatcaggtc ccggacccca cccctatgga actagaggcc 10320 ctgcagttgc ctgtgtcaga cttgcaaggg cttgacttgc agcccccagt gtccccaccg 10380 gatcaagtgg tgagtagact gagaggtggg caaagcttcc tgggtgggtg tacctgcagt 10440 gccaactgcc aggctgttaa ttcagtagga cactgtcccc aactggccca actgcacatc 10500 ctgtagtcag gaggcacagg cagaaaaatc ccaaattcaa ggcttgctcc cgttatgtaa 10560 tgagatcctg tcttggagta aaaaacaaag aagagaacta gggatagctc agaggtagat 10620 gctctcctgg catgggggtg gggtcagaaa gcaacaccaa ccggggcctg ggagggaggg 10680 actgccaacc acctggagga gtctggggta gacttggtga acaaagttca gaggccatca 10740 ggtgggatgc tggtttctta aaagccacag ataggtgggt agcattggaa agaggagtgg 10800 ggggttgcag aaagtgacaa gacacaaact ggggaggcct aagggtaaag ccagggttgt 10860 ctgaagcact gtggagctgg gaggaacacg ctaaacttct gacttcagcc cttcagttcc 10920 cctgttgact acactgtccc cagggaccca gggatgggga gaggtggacg ggggagggaa 10980 gtacgggact gatccagctc caggtcccaa ctctgatccc caccgacagg ctgaagaggc 11040 tgacctagtg gctgtccctg cccctgtggc tgaggcagag accacggtaa cgctgcagca 11100 gctccaggaa gccctggaag aagaggttct cacccggcag agcctgagcc gcgagctgga 11160 ggccatccgg accgccaacc agaacttctc caggtcaggg tcacagtgct ggggtgaggg 11220 gagaggagag cagcaaccct cgcagtctcc tcaccgatag gtcggctcac tcccctatct 11280 ttcccagcca actacaggag gccgaggtcc gaaaccgaga cctggaggcg catgttcggc 11340 agctacagga acggatggag atgctgcagg ccccaggagc cgcaggcgag tccctcacct 11400 gcttccagcc aagggggcac tgggtggaga tggggggcat gttgggtgtg tgaaccctcg 11460 gggcagggga ggagtccagg ctggggcacc gcagccgcgc cactgccttt ctcctccatc 11520 ctccacactc catacacctc tctcttctcc ttccagccat cacgggggtc cccagtcccc 11580 gggccacgga tccaccttcc catgtaagac ccctctctcc cctccccgat ccccatctta 11640 gatatgctac ccacagccct tctcccgtcc acgtttaggg tccattctcc ttgggggttc 11700 cagaagaaag ctgcccttca ctcatccatt cagcatgcac tatctaccag ctctccctcg 11760 tttcaggctt ctcgccaaat cctccccaag ggaactccct atactcccgt tctggcctcg 11820 actagattcc cgcactgcct ctcgccctgc tgctgggctc cgatcgggtc acctgtccct 11880 tctctctcca gctagatggc cccccggccg tggctgtggg ccagtgcccg ctggtggggc 11940 caggccccat gcaccgccgt cacctgctgc tccctgccag ggtatgtccc acgtccgccc 12000 accacgggcc tctgcctagc tctgcccact gagtgtcacc actgcttgct gtgcctctgt 12060 ggagctcggc ccaccgcagg gagggggggt attcgggcgg ccaatcaaca caggctgctg 12120 ctaagtagcc aatgacgagt tccaacagga gctctttctt gcgagcagac caactttagc 12180 tgcgggctgt ggggaccaga gatgcgctca gaggcccatc tatgggtata ggctgggcgg 12240 ctcccaggag ccagtgggcc cctgtagcct agtgctaatc caaccttctc tcctgcagat 12300 ccctaggcct ggcctatccg aggcgcgttg cctgctcctg ttcgccgctg ctctggctgc 12360 tgccgccaca ctgggctgca ctgggttggt ggcctatacc ggcggtctca ccccagtctg 12420 gtgtttcccg ggagccacct tcgccccctg aaccctaaga ctccaagcca tctttcattt 12480 aggcctccta ggaaggtcga gcgaccaggg agcgacccaa agcgtctctg tgcccatcgc 12540 gccccccccc cccccccacc gctccgctcc acacttctgt gagcctgggt ccccacccag 12600 ctccgctcct gtgatccagg cctgccacct ggcggccggg gagggaggaa cagggctcgt 12660 gcccagcacc cctggttcct gcagagctgg tagccaccgc tgctgcagca gctgggcatt 12720 cgccgacctt gctttactca gccccgacgt ggatgggcaa actgctcagc tcatccgatt 12780 tcactttttc actctcccag ccatcagtta caagccataa gcatgagccc cctatttcca 12840 gggacatccc attcccatag tgatggatca gcaagacctc tgccagcaca cacggagtct 12900 ttggcttcgg acagcctcac tcctgggggt tgctgcaact ccttccccgt gtacacgtct 12960 gcactctaac aacggagcca cagctgcact cccccctccc ccaaagcagt gtgggtattt 13020 attgatcttg ttatctgact cactgacaga ctccgggacc cacgttttag atgcattgag 13080 actcgacatt cctcggtatt tattgtctgt ccccacctac gacctccact cccgaccctt 13140 gcgaataaaa tacttctggt ctgccctaaa tcccgcgcaa tatctctgtt gtggaaagga 13200 aaccgccccg caggccaatg gagagtccaa tagagacaac caatggcttg agtgggagct 13260 agaggggagg caaagcgcac gaatcaggtt gaagggtggg gcttaggcat ccagccagta 13320 ggagagaagc aacaagccac cagagacacc accgcccccc accctccccc ccagctgtga 13380 cccagctgtg ccactcaagt ttggaaaaaa gtagggggtt gggccagcag cgggcacacc 13440 atcttcccac tgcgcctgcg caagccacgc gcatccgctt tttggaccga cactccagaa 13500 aagttgctgc aaactttcta gcgcgattcc ccgcccctcc tcccagctag atccaccgcc 13560 tacccgcggg gccgggaatt ccgaggggcg gagcacggcg cggagatggg aagggagggg 13620 gcccttcaag ggacccggga gatgggagcg gcttcgcgcc cttaaccctc cggacggccc 13680 attaccttct ccgttgctct gatagggaaa ctgaggccct gagtcagagg cacacaaggg 13740 gggaaggcca aaagcgcggc cagagacgga gggaaaacaa agaatcctga cagcccggga 13800 ggggggcgga cacacaggga caaggacaga cccgagtgca gagctgggtc tagtctttgg 13860 gagggggcca gaagactgca aggggaccgg gggggggggc ggcgaggagg actgggcgga 13920 ggagggggct ggggaagccc gcgggaggcg gcaaaggagg gaggaacttt ccaaagttgc 13980 caaacatggc tacctcgcct gcggagccga gcgcggggcc cgcggctcgg ggggaggcgg 14040 cggcggcgac cgaggagcag gaggaggaag cgcgccagct tctgcagact ctgcaggcag 14 100 ccgaggggga ggcggcggcg gccggggcgg gagatgcggc ggcggcggcg gactctgggt 14160 ccccgagtgg cccggggtct ccccgggaga ccgtgaccga ggtgcccact ggccttcgct 14220 tctcgcccga acaggtggca tgcgtgtgcg aggcgctgct gcaggcgggc cacgccggcc 14280 gcttgagccg cttcctgggc gcgctgcccc cggccgagcg cctacgtggc agcgatccgg 14340 tgctgcgcgc gcgggcccta gtggccttcc agcggggtga atacgccgag ctctaccaac 14400 ttctcgagag ccgccctttc cccgccgccc accacgcctt cctgcaggac ctctacctgc 14460 gcgcgcgcta ccacgaggcc gagcgggccc gtggccgtgc gctgggcgct gtggacaaat 14520 accggctgcg caagaagttc cctctgccca agaccatctg ggatggcgag gagaccgtct 14580 attgcttcaa ggagcgctcg cgagcggcgc tcaaggcctg ctaccgcggc aaccgctatc 14640 ccacgcctga cgagaagcgc cgcctggcca cgctcaccgg cctctcgctt acacaggtca 14700 gcaactggtt caagaaccgg cgacagcgcg accgcactgg gaccggcggt ggagcgcctt 14760 gcaaaaggtg aggggggaac cgaccctcct tcctcggtgg ccgctggagt ctgcgcaagt 14820 gacccttcac atccctcttc ggtggcgtcg gcgagtgcat aggctgagcg tggagagacc 14880 aggcacaccc tgggttctct gggcatcact gcctcagggg cagaggttgt tccagctact 14940 tctaagctgg gaacgcagtg ccaggaatgg gggggggggc gggggcggga cgggcagtga 15000 <210> 4 <211> 1896 <212> DNA <213> Mus musculus <400> 4 atgtcagccg aagtgcggct gaggcagctc cagcagctgg tgctggaccc aggcttcctg 60 ggactggagc ccctgctcga ccttctcctg ggcgtccacc aggagctggg tgcctctcac 120 ctagcccagg acaagtatgt ggccgacttc ttgcagtggg tggagcccat tgcagcaagg 180 cttaaggagg tccgactgca gagggatgat tttgagattt tgaaggtgat cgggcgtggg 240 gcgttcagcg aggtagcggt ggtgaagatg aaacagacgg gccaagtgta tgccatgaag 300 attatgaata agtgggacat gctgaagaga ggcgaggtgt cgtgcttccg ggaagaaagg 360 gatgtattag tgaaagggga ccggcgctgg atcacacagc tgcactttgc cttccaggat 420 gagaactacc tgtacctggt catggaatac tacgtgggcg gggacctgct aacgctgctg 480 agcaagtttg gggagcggat ccccgccgag atggctcgct tctacctggc cgagattgtc 540 atggccatag actccgtgca ccggctgggc tacgtgcaca gggacatcaa accagataac 600 attctgctgg accgatgtgg gcacattcgc ctggcagact tcggctcctg cctcaaactg 660 cagcctgatg gaatggtgag gtcgctggtg gctgtgggca ccccggacta cctgtctcct 720 gagattctgc aggccgttgg tggagggcct ggggcaggca gctacgggcc agagtgtgac 780 tggtgggcac tgggcgtgtt cgcctatgag atgttctatg ggcagacccc cttctacgcg 840 gactccacag ccgagacata tgccaagatt gtgcactaca gggaacactt gtcgctgccg 900 ctggcagaca cagttgtccc cgaggaagct caggacctca ttcgtgggct gctgtgtcct 960 gctgagataa ggctaggtcg aggtggggca ggtgatttcc agaaacatcc tttcttcttt 1020 ggccttgatt gggagggtct ccgagacagt gtacccccct ttacaccaga cttcgagggt 1080 gccacggaca catgcaattt cgatgtggtg gaggaccggc tcactgccat ggtgagcggg 1140 ggcggggaga cgctgtcaga catgcaggaa gacatgcccc ttggggtgcg cctgcccttc 1200 gtgggctact cctactgctg catggccttc agagacaatc aggtcccgga ccccacccct 1260 atggaactag aggccctgca gttgcctgtg tcagacttgc aagggcttga cttgcagccc 1320 ccagtgtccc caccggatca agtggctgaa gaggccgacc tagtggctgt ccctgcccct 1380 gtggctgagg cagagaccac ggtaacgctg cagcagctcc aggaagccct ggaagaagag 1440 gttctcaccc ggcagagcct gagccgcgag ctggaggcca tccggaccgc caaccagaac 1500 ttctccagcc aactacagga ggccgaggtc cgaaaccgag acctggaggc gcatgttcgg 1560 cagctacagg aacggatgga gatgctgcag gccccaggag ccgcagccat cacgggggtc 1620 cccagtcccc gggccacgga tccaccttcc catctagatg gccccccggc cgtggctgtg 1680 ggccagtgcc cgctggtggg gccaggcccc atgcaccgcc gtcacctgct gctccctgcc 1740 aggatcccta ggcctggcct atccgaggcg cgttgcctgc tcctgttcgc cgctgctctg 1800 gctgctgccg ccacactggg ctgcactggg ttggtggcct ataccggcgg tctcacccca 1860 gtctggtgtt tcccgggagc caccttcgcc ccctga 1896 <210> 5 <211> 771 <212> DNA <213> Mus musculus <220> <221> misc_feature <222> 89, 238, 506 <223> n = A,T,C or G <400> 5 cctgcccctg tggctgaggc agagaccacg gtaacgctgc agcagctcca ggaagccctg 60 gaagaagagg ttctcacccg gcagagctng agccgcgagc tggaggccat ccggaccgcc 120 aaccagaact tctccagcca actacaggag gccgaggtcc gaaaccgaga cctggaggcg 180 catgttcggc agctacagga acggatggag atgctgcagg ccccaggagc cgccggantc 240 cctcacctgc ttccagccaa gggggcactg ggtggagatg gggggcatgt tgggtgtgtg 300 aaccctcggg gcaggggagg agtccaggct ggggcaccgc gccgcgccac tgcctttctc 360 ctccatcctc cacactccat acacctctct cttctccttc cagccatcac gggggtccca 420 gtccccgggc cacggatcca ccttcccatc tagatggccc cccggcggtg gctgtgggcc 480 agtgcccgct ggtggggcca ggacantgtc accgccgtca cctgctgctc cctgccagga 540 ttcctaggcc tggctatccg aggcgcgttg ctgctcctgt tcgccgctgc tctggctgct 600 gcgccacact gggctgcact gggttggttg gctataccgg cggtcttcac ccagtctggt 660 gtttcccgtg agcacccttc gcccctgaaa cctaagactt caagccatct ttcatttagg 720 ccttctagga aggtcgagcg acaggggagc gacccaaagc gtctctgtgc c 771 <210> 6 <211> 434 <212> DNA <213> Mus musculus <400> 6 gagagaccca aggggtagtc agggacgggc agacatgcag ctagggttct ggggcctgga 60 caggggcagc caggccctgt gacgggaaga ccccgagctc cggcccgggg aggggccatg 120 gtgttgcctg cccaacatgt cagccgaagt gcggctgagg cagctccagc agctggtgct 180 ggacccaggc ttcctgggac tggagcccct gctcgacctt ctcctgggcg tccaccagga 240 gctgggtgcc tctcacctag cccaggacaa gtatgtggcc gacttcttgc agtgggtgga 300 gcccattgca gcaaggctta aggaggtccg actgcagagg gatgattttg agattttgaa 360 ggtgatcggg cgtggggcgt tcagcgaggt agcggtggtg aagatgaaac acacggagtc 420 tttggcttcg gaca 434 <210> 7 <211> 2688 <212> DNA <213> Mus musculus <400> 7 ccacgcgtcc gcccacgcgt ccggggcaga catgcagcta gggttctggg gcctggacag 60 gggcagccag gccctgtgac gggaagaccc cgagctccgg cccggggagg ggccatggtg 120 ttgcctgccc aacatgtcag ccgaagtgcg gctgaggcag ctccagcagc tggtgctgga 180 cccaggcttc ctgggactgg agcccctgct cgaccttctc ctgggcgtcc accaggagct 240 gggtgcctct cacctagccc aggacaagta tgtggccgac ttcttgcagt gggtggagcc 300 cattgcagca aggcttaagg aggtccgact gcagagggat gattttgaga ttttgaaggt 360 gatcgggcgt ggggcgttca gcgaggtagc ggtggtgaag atgaaacaga cgggccaagt 420 gtatgccatg aagattatga ataagtggga catgctgaag agaggcgagg tgtcgtgctt 480 ccgggaagaa agggatgtat tagtgaaagg ggaccggcgc tggatcacac agctgcactt 540 tgccttccag gatgagaact acctgtacct ggtcatggaa tactacgtgg gcggggacct 600 gctaacgctg ctgagcaagt ttggggagcg gatccccgcc gagatggctc gcttctacct 660 ggccgagatt gtcatggcca tagactccgt gcaccggctg ggctacgtgc acagggacat 720 caaaccagat aacattctgc tggaccgatg tgggcacatt cgcctggcag acttcggctc 780 ctgcctcaaa ctgcagcctg atggaatggt gaggtcgctg gtggctgtgg gcaccccgga 840 ctacctgtct cctgagattc tgcaggccgt tggtggaggg cctggggcag gcagctacgg 900 gccagagtgt gactggtggg cactgggcgt gttcacctat gagatgttct atgggcagac 960 ccccttctac gcggactcca cagccgagac atatgccaag attgtgcact acagggaaca 1020 cttgtcgctg ccgctggcag acacagttgt ccccgaggaa gctcaggacc tcattcgtgg 1080 gctgctgtgt cctgctgaga taaggctagg tcgaggtggg gcaggtgatt tccagaaaca 1140 tcctttcttc tttggccttg attgggaggg tctccgagac agtgtacccc cctttacacc 1200 agacttcgag ggtgccacgg acacatgcaa tttcgatgtg gtggaggacc ggctcactgc 1260 catggtgagc gggggcgggg agacgctgtc agacatgcag gaagacatgc cccttggggt 1320 gcgcctgccc ttcgtgggct actcctactg ctgcatggcc ttcagagaca atcaggtccc 1380 ggaccccacc cctatggaac tagaggccct gcagttgcct gtgtcagact tgcaagggct 1440 tgacttgcag cccccagtgt ccccaccgga tcaagtggct gaagaggctg acctagtggc 1500 tgtccctgcc cctgtggctg aggcagagac cacggtaacg ctgcagcagc tccaggaagc 1560 cctggaagaa gaggttctca cccggcagag cctgagccgc gagctggagg ccatccggac 1620 cgccaaccag aacttctcca gccaactaca ggaggccgag gtccgaaacc gagacctgga 1680 ggcgcatgtt cggcagctac aggaacggat ggagatgctg caggccccag gagccgcaga 1740 tccctaggcc tggcctatcc gaggcgcgtt gcctgctcct gttcgccgct gctctggctg 1800 ctgccgccac actgggctgc actgggttgg tggcctatac cggcggtctc accccagtct 1860 ggtgtttccc gggagccacc ttcgccccct gaaccctaag actccaagcc atctttcatt 1920 taggcctcct aggaaggtcg agcgaccagg gagcgaccca aagcgtctct gtgcccatcg 1980 cccccccccc cccccccacc gctccgctcc acacttctgt gagcctgggt ccccacccag 2040 ctccgctcct gtgatccagg cctgccacct ggcggccggg gagggaggaa cagggctcgt 2100 gcccagcacc cctggttcct gcagagctgg tagccaccgc tgctgcagca gctgggcatt 2160 cgccgacctt gctttactca gccccgacgt ggatgggcaa actgctcagc tcatccgatt 2220 tcactttttc actctcccag ccatcagtta caagccataa gcatgagccc cctatttcca 2280 gggacatccc attcccatag tgatggatca gcaagacctc tgccagcaca cacggagtct 2340 ttggcttcgg acagcctcac tcctgggggt tgctgcaact ccttccccgt gtacacgtct 2400 gcactctaac aacggagcca cagctgcact cccccctccc ccaaagcagt gtgggtattt 2460 attgatcttg ttatctgact cactgacaga ctccgggacc cacgttttag atgcattgag 2520 actcgacatt cctcggtatt tattgtctgt ccccacctac gacctccact cccgaccctt 2580 gcgaataaaa tacttctggt ctgccctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2688 <210> 8 <211> 2862 <212> DNA <213> Mus musculus <400> 8 gggatagtca gggacgggca gacatgcagc tagggttctg gggcctggac aggggcagcc 60 aggccctgtg acgggaagac cccgagctcc ggcccgggga ggggccatgg tgttgcctgc 120 ccaacatgtc agccgaagtg cggctgaggc agctccagca gctggtgctg gacccaggct 180 tcctgggact ggagcccctg ctcgaccttc tcctgggcgt ccaccaggag ctgggtgcct 240 ctcacctagc ccaggacaag tatgtggccg acttcttgca gtgggtggag cccattgcag 300 caaggcttaa ggaggtccga ctgcagaggg atgattttga gattttgaag gtgatcgggc 360 gtggggcgtt cagcgaggta gcggtggtga agatgaaaca gacgggccaa gtgtatgcca 420 tgaagattac gaataagtgg gacatgctga agagaggcga ggtgtcgtgc ttccgggaag 480 aaagggatgt attagtgaaa ggggaccggc gctggatcac acagctgcac tttgccttcc 540 aggatgagaa ctacctgtac ctggtcatgg aatactacgt gggcggggac ctgctaacgc 600 tgctgagcaa gtttggggag cggatccccg ccgagatggc tcgcttctac ctggccgaga 660 ttgtcatggc catagactcc gtgcaccggc tgggctacgt gcacagggac atcaaaccag 720 ataacattct gctggaccga tgtgggcaca ttcgcctggc agacttcggc tcctgcctca 780 aactgcagcc tgatggaatg gtgaggtcgc tggtggctgt gggcaccccg gactacctgt 840 ctcctgagat tctgcaggcc gttggtggag ggcctggggc aggcagctac gggccagagt 900 gtgactggtg ggcactgggc gtgttcacct atgagatgtt ctatgggcag acccccttct 960 acgcggactc cacagccgag acatatgcca agattgtgca ctacagggaa cacttgtcgc 1020 tgccgctggc agacacagtt gtccccgagg aagctcagga cctcattcgt gggctgctgt 1080 gtcctgctga gataaggcta ggtcgaggtg gggcaggtga tttccagaaa catcctttct 1140 tctttggcct tgattgggag ggtctccgag acagtgtacc cccctttaca ccagacttcg 1200 agggtgccac ggacacatgc aatttcgatg tggtggagga ccggctcact gccatggaga 1260 cgctgtcaga catgcaggaa gacatgcccc ttggggtgcg cctgcccttc gtgggctact 1320 cctactgctg catggccttc agagacaatc aggtcccgga ccccacccct atggaactag 1380 aggccctgca gttgcctgtg tcagacttgc aagggcttga cttgcagccc ccagtgtccc 1440 caccggatca agtggctgaa gaggctgacc tagtggctgt ccctgcccct gtggctgagg 1500 cagagaccac ggtaacgctg cagcagctcc aggaagccct ggaagaagag gttctcaccc 1560 ggcagagcct gagccgcgag ctggaggcca tccggaccgc caaccagaac ttctccagcc 1620 aactacagga ggccgaggtc cgaaaccgag acctggaggc gcatgttcgg cagctacagg 1680 aacggatgga gatgctgcag gccccaggag ccgcagccat cacgggggtc cccagtcccc 1740 gggccacgga tccaccttcc catgcttctc gccaaatcct ccccaaggga actccctaga 1800 ctcccgttct ggcctcgact agattcccgc actgcctctc gccctgctgc tgggctccga 1860 tcgggtcacc tgtcccttct ctctccagct agatggcccc ccggccgtgg ctgtgggcca 1920 gtgcccgctg gtggggccag gccccatgca ccgccgtcac ctgctgctcc ctgccaggat 1980 ccctaggcct ggcctatccg aggcgcgttg cctgctcctg ttcgccgctg ctctggctgc 2040 tgccgccaca ctgggctgca ctgggttggt ggcctatacc ggcggtctca ccccagtctg 2100 gtgtttcccg ggagccacct tcgccccctg aaccctaaga ctccaagcca tctttcattt 2160 aggcctccta ggaaggtcga gcgaccaggg agcgacccaa agcgtctctg tgcccatcgc 2220 cccccccccc ccccccaccg ctccgctcca cacttctgtg agcctgggtc cccacccagc 2280 tccgctcctg tgatccaggc ctgccacctg gcggccgggg agggaggaac agggctcgtg 2340 cccagcaccc ctggttcctg cagagctggt agccaccgct gctgcagcag ctgggcattc 2400 gccgaccttg ctttactcag ccccgacgtg gatgggcaaa ctgctcagct catccgattt 2460 cactttttca ctctcccagc catcagttac aagccataag catgagcccc ctatttccag 2520 ggacatccca ttcccatagt gatggatcag caagacctct gccagcacac acggagtctt 2580 tggcttcgga cagcctcact cctgggggtt gctgcaactc cttccccgtg tacacgtctg 2640 cactctaaca acggagccac agctgcactc ccccctcccc caaagcagtg tgggtattta 2700 ttgatcttgt tatctgactc actgacagac tccgggaccc acgttttaga tgcattgaga 2760 ctcgacattc ctcggtattt attgtctgtc cccacctacg acctccactc ccgacccttg 2820 cgaataaaat acttctggtc tgccctaaaa aaaaaaaaaa aa 2862 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 agcctgagcc gggagatg 18 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 gcgtagttga ctggcgaagt t 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 11 aggccatccg cacggacaac c 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 12 ctggctgcat gtctgcctgt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 13 ccaggagaag gtcgagcagg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 14 tctatggcca tgacaatctc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 15 atgtccctgt gcacgtagcc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 16 atgtgtccgg aagtcgcctg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 17 ctcaggctct gccgggtgag 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 18 ggcactggcc cacagccacg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 19 cctggccgaa agaaagaaat 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 20 aaagaaatgg tctgtgatcc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 21 aagaaagaaa tggtctgtga 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 22 ggccgaaaga aagaaatggt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 23 cctcagcctg gccgaaagaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 24 gggcctcagc ctggccgaaa 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 25 tcagggcctc agcctggccg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 26 ctgcagtttg cccatccacg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 27 ggcctgcagt ttgcccatcc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 28 ccaggcctgc agtttgccca 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 29 gccttcccag gcctgcagtt 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 30 gctgccttcc caggcctgca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 31 cttgctgcct tcccaggcct 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 32 gcccggcttg ctgccttccc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 33 acggcccggc ttgctgcctt 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 34 cggacggccc ggcttgctgc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 35 acacggacgg cccggcttgc 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 36 gatggaacac ggacggcccg 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 37 gaggatggaa cacggacggc 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 38 gtggaggatg gaacacggac 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 39 gcgaaccaac gataggtggg 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 40 tttgcgaacc aacgataggt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 41 ttgcactttg cgaaccaacg 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 42 gctttgcact ttgcgaacca 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 43 aaagctttgc actttgcgaa 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 44 aagaaagctt tgcactttgc 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 45 cacaagaaag ctttgcactt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 46 gtcatgcaca agaaagcttt 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 47 acgctcccca gagcagggcg 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 48 gcagagatcg cgccagacgc 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 49 caggcagaga tcgcgccaga 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 50 aagcaggcag agatcgcgcc 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 51 ccgagtaagc aggcagagat 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 52 ttcccgagta agcaggcaga 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 53 gcaaatttcc cgagtaagca 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 54 aaagcaaatt tcccgagtaa 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 55 ttggcaaaag caaatttccc 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 56 ggtttggcaa aagcaaattt 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 57 gcgggtttgg caaaagcaaa 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 58 aaagcgggtt tggcaaaagc 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 59 cccgaaaaag cgggtttggc 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 60 atccccgaaa aagcgggttt 20 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 61 cgggatcccc gaaaaagcgg 20 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 62 gcgcgggatc cccgaaaaag 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 63 gagagcagcg caagtgagga 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 64 tccgagagca gcgcaagtga 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 65 ggctccgaga gcagcgcaag 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 66 aagcgggcgg agccggctgg 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 67 ccgaagcggg cggagccggc 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 68 aaaccgccga agcgggcgga 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 69 tccaaaccgc cgaagcgggc 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 70 atatccaaac cgccgaagcg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 71 taaatatcca aaccgccgaa 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 72 caataaatat ccaaaccgcc 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 73 cgaggtcaat aaatatccaa 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 74 ggacgaggtc aataaatatc 20 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 75 ggaggacgag gtcaataaat 20 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 76 gtcggaggac gaggtcaata 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 77 cgagtcggag gacgaggtca 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 78 tgtcagcgag tcggaggacg 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 79 gcctgtcagc gagtcggagg 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 80 gtagcctgtc agcgagtcgg 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 81 cctgtagcct gtcagcgagt 20 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 82 ggtcctgtag cctgtcagcg 20 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 83 aaataccgag gaatgtcggg 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 84 aataaatacc gaggaatgtc 20 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 85 gacaataaat accgaggaat 20 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 86 cggggccccg gagtcgaaga 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 87 ccaacggggc cccggagtcg 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 88 ttccaacggg gccccggagt 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 89 gtcttccaac ggggccccgg 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 90 cagtcttcca acggggcccc 20 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 91 ctcagtcttc caacggggcc 20 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 92 gcactcagtc ttccaacggg 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 93 ccccgggcac tcagtcttcc 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 94 tgccccgggc actcagtctt 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 95 cgtgccccgg gcactcagtc 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 96 gtgccgtgcc ccgggcactc 20 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 97 tctgtgccgt gccccgggca 20 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 98 gcttctgtgc cgtgccccgg 20 <210> 99 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 99 gcggcttctg tgccgtgccc 20 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 100 gcgcggcttc tgtgccgtgc 20 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 101 gggcgcggct tctgtgccgt 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 102 ggcggtgggc gcggcttctg 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 103 ggcaggcggt gggcgcggct 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 104 ctggcaggcg gtgggcgcgg 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 105 aactggcagg cggtgggcgc 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 106 gtgaactggc aggcggtggg 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 107 ggttgtgaac tggcaggcgg 20 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 108 gcggttgtga actggcaggc 20 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 109 cggagcggtt gtgaactggc 20 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 110 cgctcggagc ggttgtgaac 20 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 111 cccacgctcg gagcggttgt 20 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 112 agacccacgc tcggagcggt 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 113 cggagaccca cgctcggagc 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 114 gggcggagac ccacgctcgg 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 115 gctgggcgga gacccacgct 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 116 ggagctgggc ggagacccac 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 117 ctggagctgg gcggagaccc 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 118 ggactggagc tgggcggaga 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 119 caggactgga gctgggcgga 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 120 atcacaggac tggagctggg 20 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 121 gggcgggccc ggatcacagg 20 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 122 gggggcgggc ccggatcaca 20 <210> 123 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 123 aggcagcacc atggcccctc 20 <210> 124 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 124 ggtccaacac cagctgctgg 20 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 125 cgatcacctt cagaatctcg 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 126 cttgttcatg atcttcatgg 20 <210> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 127 ccccattcac caacacgtcc 20 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 128 gcgtgatcca ccgccggtcc 20 <210> 129 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 129 gtaatactcc atgaccaggt 20 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 130 gcagtgtcag caggtccccg 20 <210> 131 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 131 caccgagtct atggccatga 20 <210> 132 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 132 acgtagccaa gccggtgcac 20 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 133 atgtggccac agcggtccag 20 <210> 134 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 134 cttcgtccac cagcggcaga 20 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 135 gaccccttcg tccaccagcg 20 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 136 cctgctccac cccggcccag 20 <210> 137 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 137 cggaagtcgc ctgctccacc 20 <210> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 138 cggagaccat cccagtcgag 20 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 139 tgagggccat gcaggagtag 20 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 140 ctccagttcc atgggtgtgg 20 <210> 141 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 141 gcgcttgcac gtgtggctca 20 <210> 142 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 142 gccacttcag ctgtttcatc 20 <210> 143 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 143 gcctcagcct ctgccgcagg 20 <210> 144 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 144 gcagcgtcac ctcggcctca 20 <210> 145 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 145 ggctcaggct ctgccgggtg 20 <210> 146 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 146 ttccgagcct ctgcctcgcg 20 <210> 147 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 147 ggtcccggtt ccgagcctct 20 <210> 148 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 148 atccgctcct gcaactgccg 20 <210> 149 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 149 gcaactccat ccgctcctgc 20 <210> 150 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 150 aggtggatcc gtggcccggg 20 <210> 151 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 151 cgcggcttct gtgccgtgcc 20 <210> 152 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 152 ttgctgcctt cccaggcctg 20 <210> 153 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 153 tgctcccgac aagctccaga 20 <210> 154 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 154 agaacctgcc cattgctgaa 20 <210> 155 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 155 cactgagggc cagacatatg 20 <210> 156 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 156 ctctagattc agatgcaggt 20 <210> 157 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 157 cgggccgtcc gtgtt 15 <210> 158 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 158 ctttgcactt tgcgaaccaa 20 <210> 159 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 159 catcctccac gcacccccac c 21 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 160 gcctggcagc ccctgtccag 20 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 161 ggcctggcag cccctgtcca 20 <210> 162 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 162 gggcctggca gcccctgtcc 20 <210> 163 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 163 atggcccctc cccgggccgg 20 <210> 164 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 164 catggcccct ccccgggccg 20 <210> 165 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 165 ccatggcccc tccccgggcc 20 <210> 166 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 166 accatggccc ctccccgggc 20 <210> 167 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 167 caccatggcc cctccccggg 20 <210> 168 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 168 gcaccatggc ccctccccgg 20 <210> 169 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 169 agcaccatgg cccctccccg 20 <210> 170 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 170 cagcaccatg gcccctcccc 20 <210> 171 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 171 gcagcaccat ggcccctccc 20 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 172 ggcagcacca tggcccctcc 20 <210> 173 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 173 caggcagcac catggcccct 20 <210> 174 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 174 acaggcagca ccatggcccc 20 <210> 175 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 175 ggacaggcag caccatggcc 20 <210> 176 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 176 tggacaggca gcaccatggc 20 <210> 177 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 177 ttggacaggc agcaccatgg 20 <210> 178 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 178 gttggacagg cagcaccatg 20 <210> 179 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 179 tgttggacag gcagcaccat 20 <210> 180 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 180 atgttggaca ggcagcacca 20 <210> 181 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 181 catgttggac aggcagcacc 20 <210> 182 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 182 acatgttgga caggcagcac 20 <210> 183 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 183 gacatgttgg acaggcagca 20 <210> 184 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 184 tgacatgttg gacaggcagc 20 <210> 185 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 185 ctgacatgtt ggacaggcag 20 <210> 186 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 186 gctgacatgt tggacaggca 20 <210> 187 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 187 ggctgacatg ttggacaggc 20 <210> 188 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 188 cggctgacat gttggacagg 20 <210> 189 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 189 tcggctgaca tgttggacag 20 <210> 190 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 190 ctcggctgac atgttggaca 20 <210> 191 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 191 cctcggctga catgttggac 20 <210> 192 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 192 acctcggctg acatgttgga 20 <210> 193 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 193 cacctcggct gacatgttgg 20 <210> 194 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 194 gcacctcggc tgacatgttg 20 <210> 195 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 195 cgcacctcgg ctgacatgtt 20 <210> 196 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 196 ccgcacctcg gctgacatgt 20 <210> 197 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 197 gccgcacctc ggctgacatg 20 <210> 198 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 198 agccgcacct cggctgacat 20 <210> 199 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 199 cagccgcacc tcggctgaca 20 <210> 200 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 200 tcagccgcac ctcggctgac 20 <210> 201 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 201 ctcagccgca cctcggctga 20 <210> 202 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 202 cctcagccgc acctcggctg 20 <210> 203 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 203 gcctcagccg cacctcggct 20 <210> 204 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 204 ccaacaccag ctgctggagc 20 <210> 205 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 205 tccaacacca gctgctggag 20 <210> 206 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 206 gtccaacacc agctgctgga 20 <210> 207 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 207 gggtccaaca ccagctgctg 20 <210> 208 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 208 ggctccagcc ccaggaagcc 20 <210> 209 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 209 gggctccagc cccaggaagc 20 <210> 210 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 210 caggagaagg tcgagcaggg 20 <210> 211 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 211 cccaggagaa ggtcgagcag 20 <210> 212 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 212 gcccaggaga aggtcgagca 20 <210> 213 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 213 cgcccaggag aaggtcgagc 20 <210> 214 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 214 acgcccagga gaaggtcgag 20 <210> 215 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 215 tcctgggcca gttcggaggc 20 <210> 216 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 216 gtcctgggcc agttcggagg 20 <210> 217 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 217 tgtcctgggc cagttcggag 20 <210> 218 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 218 ttgtcctggg ccagttcgga 20 <210> 219 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 219 cttgtcctgg gccagttcgg 20 <210> 220 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 220 acttgtcctg ggccagttcg 20 <210> 221 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 221 tacttgtcct gggccagttc 20 <210> 222 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 222 gtacttgtcc tgggccagtt 20 <210> 223 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 223 cgtacttgtc ctgggccagt 20 <210> 224 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 224 actgcaagaa gtcggccacg 20 <210> 225 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 225 ccactgcaag aagtcggcca 20 <210> 226 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 226 cccactgcaa gaagtcggcc 20 <210> 227 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 227 gcccactgca agaagtcggc 20 <210> 228 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 228 cgcccactgc aagaagtcgg 20 <210> 229 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 229 ccgcccactg caagaagtcg 20 <210> 230 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 230 tccgcccact gcaagaagtc 20 <210> 231 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 231 ctccgcccac tgcaagaagt 20 <210> 232 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 232 gctccgccca ctgcaagaag 20 <210> 233 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 233 ggctccgccc actgcaagaa 20 <210> 234 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 234 gggctccgcc cactgcaaga 20 <210> 235 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 235 tgggctccgc ccactgcaag 20 <210> 236 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 236 atgggctccg cccactgcaa 20 <210> 237 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 237 gatgggctcc gcccactgca 20 <210> 238 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 238 cgatgggctc cgcccactgc 20 <210> 239 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 239 acgatgggct ccgcccactg 20 <210> 240 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 240 cacgatgggc tccgcccact 20 <210> 241 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 241 ccacgatggg ctccgcccac 20 <210> 242 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 242 accacgatgg gctccgccca 20 <210> 243 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 243 caccacgatg ggctccgccc 20 <210> 244 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 244 tcaccacgat gggctccgcc 20 <210> 245 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 245 ctcaccacga tgggctccgc 20 <210> 246 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 246 cctcaccacg atgggctccg 20 <210> 247 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 247 gcctcaccac gatgggctcc 20 <210> 248 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 248 agcctcacca cgatgggctc 20 <210> 249 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 249 aagcctcacc acgatgggct 20 <210> 250 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 250 taagcctcac cacgatgggc 20 <210> 251 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 251 ttaagcctca ccacgatggg 20 <210> 252 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 252 cttaagcctc accacgatgg 20 <210> 253 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 253 ccttaagcct caccacgatg 20 <210> 254 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 254 tccttaagcc tcaccacgat 20 <210> 255 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 255 ctccttaagc ctcaccacga 20 <210> 256 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 256 cctccttaag cctcaccacg 20 <210> 257 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 257 acctccttaa gcctcaccac 20 <210> 258 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 258 gacctcctta agcctcacca 20 <210> 259 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 259 ggacctcctt aagcctcacc 20 <210> 260 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 260 cggacctcct taagcctcac 20 <210> 261 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 261 tcggacctcc ttaagcctca 20 <210> 262 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 262 gtcggacctc cttaagcctc 20 <210> 263 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 263 cagtcggacc tccttaagcc 20 <210> 264 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 264 gcagtcggac ctccttaagc 20 <210> 265 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 265 tgcagtcgga cctccttaag 20 <210> 266 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 266 ccttcagaat ctcgaagtcg 20 <210> 267 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 267 accttcagaa tctcgaagtc 20 <210> 268 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 268 tcaccttcag aatctcgaag 20 <210> 269 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 269 atcaccttca gaatctcgaa 20 <210> 270 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 270 gatcaccttc agaatctcga 20 <210> 271 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 271 ccgatcacct tcagaatctc 20 <210> 272 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 272 tccgatcacc ttcagaatct 20 <210> 273 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 273 gtccgatcac cttcagaatc 20 <210> 274 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 274 cgtccgatca ccttcagaat 20 <210> 275 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 275 cccgtctgct tcatcttcac 20 <210> 276 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 276 gcccgtctgc ttcatcttca 20 <210> 277 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 277 ggcccgtctg cttcatcttc 20 <210> 278 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 278 tggcccgtct gcttcatctt 20 <210> 279 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 279 ctggcccgtc tgcttcatct 20 <210> 280 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 280 cctggcccgt ctgcttcatc 20 <210> 281 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 281 acctggcccg tctgcttcat 20 <210> 282 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 282 cacctggccc gtctgcttca 20 <210> 283 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 283 acacctggcc cgtctgcttc 20 <210> 284 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 284 tacacctggc ccgtctgctt 20 <210> 285 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 285 ttgttcatga tcttcatggc 20 <210> 286 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 286 acttgttcat gatcttcatg 20 <210> 287 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 287 cacttgttca tgatcttcat 20 <210> 288 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 288 ccacttgttc atgatcttca 20 <210> 289 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 289 cccacttgtt catgatcttc 20 <210> 290 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 290 tcccacttgt tcatgatctt 20 <210> 291 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 291 gtcccacttg ttcatgatct 20 <210> 292 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 292 tgtcccactt gttcatgatc 20 <210> 293 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 293 atgtcccact tgttcatgat 20 <210> 294 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 294 catgtcccac ttgttcatga 20 <210> 295 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 295 gcatgtccca cttgttcatg 20 <210> 296 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 296 agcatgtccc acttgttcat 20 <210> 297 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 297 cagcatgtcc cacttgttca 20 <210> 298 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 298 tcagcatgtc ccacttgttc 20 <210> 299 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 299 ttcagcatgt cccacttgtt 20 <210> 300 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 300 cttcagcatg tcccacttgt 20 <210> 301 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 301 tcttcagcat gtcccacttg 20 <210> 302 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 302 cctcttcagc atgtcccact 20 <210> 303 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 303 ccctcttcag catgtcccac 20 <210> 304 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 304 cccctcttca gcatgtccca 20 <210> 305 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 305 gcccctcttc agcatgtccc 20 <210> 306 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 306 cgcccctctt cagcatgtcc 20 <210> 307 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 307 tcgcccctct tcagcatgtc 20 <210> 308 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 308 ctcgcccctc ttcagcatgt 20 <210> 309 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 309 cctcgcccct cttcagcatg 20 <210> 310 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 310 acctcgcccc tcttcagcat 20 <210> 311 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 311 cacctcgccc ctcttcagca 20 <210> 312 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 312 acacctcgcc cctcttcagc 20 <210> 313 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 313 gacacctcgc ccctcttcag 20 <210> 314 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 314 gccaggcgga tgtggccaca 20 <210> 315 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 315 accgcaccgt tccatctgcc 20 <210> 316 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 316 gaccgcaccg ttccatctgc 20 <210> 317 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 317 acagcctgca ggatctcggg 20 <210> 318 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 318 cacagcctgc aggatctcgg 20 <210> 319 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 319 ccacagcctg caggatctcg 20 <210> 320 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 320 cccacagcct gcaggatctc 20 <210> 321 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 321 gcccacagcc tgcaggatct 20 <210> 322 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 322 cgcccacagc ctgcaggatc 20 <210> 323 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 323 ccgcccacag cctgcaggat 20 <210> 324 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 324 accgcccaca gcctgcagga 20 <210> 325 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 325 caccgcccac agcctgcagg 20 <210> 326 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 326 ccaccgccca cagcctgcag 20 <210> 327 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 327 cccaccgccc acagcctgca 20 <210> 328 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 328 gcccaccgcc cacagcctgc 20 <210> 329 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 329 ggcccaccgc ccacagcctg 20 <210> 330 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 330 aggcccaccg cccacagcct 20 <210> 331 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 331 caggcccacc gcccacagcc 20 <210> 332 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 332 ccaggcccac cgcccacagc 20 <210> 333 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 333 cccaggccca ccgcccacag 20 <210> 334 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 334 tcccaggccc accgcccaca 20 <210> 335 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 335 gtcccaggcc caccgcccac 20 <210> 336 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 336 tgtcccaggc ccaccgccca 20 <210> 337 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 337 ctgtcccagg cccaccgccc 20 <210> 338 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 338 cctgtcccag gcccaccgcc 20 <210> 339 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 339 gcctgtccca ggcccaccgc 20 <210> 340 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 340 tgcctgtccc aggcccaccg 20 <210> 341 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 341 ctgcctgtcc caggcccacc 20 <210> 342 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 342 gctgcctgtc ccaggcccac 20 <210> 343 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 343 agctgcctgt cccaggccca 20 <210> 344 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 344 tagctgcctg tcccaggccc 20 <210> 345 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 345 gtagctgcct gtcccaggcc 20 <210> 346 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 346 cgtagctgcc tgtcccaggc 20 <210> 347 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 347 ccgtagctgc ctgtcccagg 20 <210> 348 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 348 cccgtagctg cctgtcccag 20 <210> 349 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 349 gcccgtagct gcctgtccca 20 <210> 350 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 350 ggcccgtagc tgcctgtccc 20 <210> 351 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 351 tagaacattt cataggcgaa 20 <210> 352 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 352 tctccgccgt ggaatccgcg 20 <210> 353 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 353 gtctccgccg tggaatccgc 20 <210> 354 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 354 ggtctccgcc gtggaatccg 20 <210> 355 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 355 aggtctccgc cgtggaatcc 20 <210> 356 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 356 taggtctccg ccgtggaatc 20 <210> 357 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 357 ttgtagtgga cgatcttgcc 20 <210> 358 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 358 cttgtagtgg acgatcttgc 20 <210> 359 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 359 ccttgtagtg gacgatcttg 20 <210> 360 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 360 tccttgtagt ggacgatctt 20 <210> 361 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 361 ctccttgtag tggacgatct 20 <210> 362 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 362 gctccttgta gtggacgatc 20 <210> 363 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 363 tgctccttgt agtggacgat 20 <210> 364 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 364 gtgctccttg tagtggacga 20 <210> 365 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 365 ggtgctcctt gtagtggacg 20 <210> 366 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 366 aggtgctcct tgtagtggac 20 <210> 367 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 367 gaggtgctcc ttgtagtgga 20 <210> 368 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 368 agaggtgctc cttgtagtgg 20 <210> 369 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 369 gagaggtgct ccttgtagtg 20 <210> 370 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 370 agagaggtgc tccttgtagt 20 <210> 371 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 371 gagagaggtg ctccttgtag 20 <210> 372 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 372 agagagaggt gctccttgta 20 <210> 373 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 373 cagagagagg tgctccttgt 20 <210> 374 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 374 ggcagagaga ggtgctcctt 20 <210> 375 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 375 cggcagagag aggtgctcct 20 <210> 376 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 376 gcggcagaga gaggtgctcc 20 <210> 377 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 377 agcggcagag agaggtgctc 20 <210> 378 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 378 cagcggcaga gagaggtgct 20 <210> 379 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 379 ccagcggcag agagaggtgc 20 <210> 380 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 380 ggcccagccg tgtctccggg 20 <210> 381 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 381 cggcccagcc gtgtctccgg 20 <210> 382 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 382 ccggcccagc cgtgtctccg 20 <210> 383 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 383 cccggcccag ccgtgtctcc 20 <210> 384 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 384 ccccggccca gccgtgtctc 20 <210> 385 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 385 accccggccc agccgtgtct 20 <210> 386 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 386 caccccggcc cagccgtgtc 20 <210> 387 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 387 ccaccccggc ccagccgtgt 20 <210> 388 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 388 tccaccccgg cccagccgtg 20 <210> 389 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 389 ctccaccccg gcccagccgt 20 <210> 390 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 390 gctccacccc ggcccagccg 20 <210> 391 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 391 tgctccaccc cggcccagcc 20 <210> 392 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 392 ctgctccacc ccggcccagc 20 <210> 393 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 393 aagggatgtg tccggaagtc 20 <210> 394 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 394 gaagggatgt gtccggaagt 20 <210> 395 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 395 agaagggatg tgtccggaag 20 <210> 396 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 396 aagaagggat gtgtccggaa 20 <210> 397 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 397 gaagaaggga tgtgtccgga 20 <210> 398 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 398 agaagaaggg atgtgtccgg 20 <210> 399 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 399 aagaagaagg gatgtgtccg 20 <210> 400 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 400 aaagaagaag ggatgtgtcc 20 <210> 401 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 401 caaagaagaa gggatgtgtc 20 <210> 402 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 402 ccaaagaaga agggatgtgt 20 <210> 403 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 403 ggccaaagaa gaagggatgt 20 <210> 404 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 404 aggccaaaga agaagggatg 20 <210> 405 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 405 gaggccaaag aagaagggat 20 <210> 406 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 406 cgaggccaaa gaagaaggga 20 <210> 407 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 407 tcgaggccaa agaagaaggg 20 <210> 408 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 408 gtcgaggcca aagaagaagg 20 <210> 409 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 409 agtcgaggcc aaagaagaag 20 <210> 410 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 410 cagtcgaggc caaagaagaa 20 <210> 411 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 411 ccagtcgagg ccaaagaaga 20 <210> 412 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 412 cccagtcgag gccaaagaag 20 <210> 413 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 413 tcccagtcga ggccaaagaa 20 <210> 414 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 414 atcccagtcg aggccaaaga 20 <210> 415 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 415 catcccagtc gaggccaaag 20 <210> 416 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 416 ccatcccagt cgaggccaaa 20 <210> 417 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 417 accatcccag tcgaggccaa 20 <210> 418 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 418 gaccatccca gtcgaggcca 20 <210> 419 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 419 agaccatccc agtcgaggcc 20 <210> 420 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 420 gagaccatcc cagtcgaggc 20 <210> 421 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 421 ggagaccatc ccagtcgagg 20 <210> 422 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 422 ttcgaaatcc ggtgtaaagg 20 <210> 423 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 423 cttcgaaatc cggtgtaaag 20 <210> 424 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 424 ccttcgaaat ccggtgtaaa 20 <210> 425 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 425 accttcgaaa tccggtgtaa 20 <210> 426 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 426 caccttcgaa atccggtgta 20 <210> 427 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 427 gcaccttcga aatccggtgt 20 <210> 428 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 428 ggcaccttcg aaatccggtg 20 <210> 429 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 429 tggcaccttc gaaatccggt 20 <210> 430 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 430 gtggcacctt cgaaatccgg 20 <210> 431 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 431 ggtggcacct tcgaaatccg 20 <210> 432 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 432 cggtggcacc ttcgaaatcc 20 <210> 433 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 433 tcggtggcac cttcgaaatc 20 <210> 434 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 434 gtcggtggca ccttcgaaat 20 <210> 435 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 435 tgtcggtggc accttcgaaa 20 <210> 436 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 436 gtgtcggtgg caccttcgaa 20 <210> 437 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 437 tgtgtcggtg gcaccttcga 20 <210> 438 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 438 atgtgtcggt ggcaccttcg 20 <210> 439 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 439 catgtgtcgg tggcaccttc 20 <210> 440 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 440 gcatgtgtcg gtggcacctt 20 <210> 441 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 441 tgcatgtgtc ggtggcacct 20 <210> 442 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 442 ttgcatgtgt cggtggcacc 20 <210> 443 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 443 gttgcatgtg tcggtggcac 20 <210> 444 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 444 agttgcatgt gtcggtggca 20 <210> 445 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 445 aagttgcatg tgtcggtggc 20 <210> 446 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 446 gaagttgcat gtgtcggtgg 20 <210> 447 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 447 cgaagttgca tgtgtcggtg 20 <210> 448 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 448 gtcgaagttg catgtgtcgg 20 <210> 449 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 449 agtcgaagtt gcatgtgtcg 20 <210> 450 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 450 aagtcgaagt tgcatgtgtc 20 <210> 451 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 451 caagtcgaag ttgcatgtgt 20 <210> 452 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 452 ccaagtcgaa gttgcatgtg 20 <210> 453 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 453 accaagtcga agttgcatgt 20 <210> 454 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 454 caccaagtcg aagttgcatg 20 <210> 455 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 455 ccaccaagtc gaagttgcat 20 <210> 456 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 456 tccaccaagt cgaagttgca 20 <210> 457 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 457 ctccaccaag tcgaagttgc 20 <210> 458 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 458 cctccaccaa gtcgaagttg 20 <210> 459 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 459 tcctccacca agtcgaagtt 20 <210> 460 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 460 gtcctccacc aagtcgaagt 20 <210> 461 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 461 cgtcctccac caagtcgaag 20 <210> 462 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 462 ccgtcctcca ccaagtcgaa 20 <210> 463 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 463 cccgtcctcc accaagtcga 20 <210> 464 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 464 gcccgtcctc caccaagtcg 20 <210> 465 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 465 agcccgtcct ccaccaagtc 20 <210> 466 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 466 gagcccgtcc tccaccaagt 20 <210> 467 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 467 tgagcccgtc ctccaccaag 20 <210> 468 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 468 ggttccgagc ctctgcctcg 20 <210> 469 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 469 cggttccgag cctctgcctc 20 <210> 470 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 470 ccggttccga gcctctgcct 20 <210> 471 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 471 cccggttccg agcctctgcc 20 <210> 472 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 472 tcccggttcc gagcctctgc 20 <210> 473 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 473 gtcccggttc cgagcctctg 20 <210> 474 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 474 aggtcccggt tccgagcctc 20 <210> 475 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 475 taggtcccgg ttccgagcct 20 <210> 476 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 476 ctaggtcccg gttccgagcc 20 <210> 477 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 477 tctaggtccc ggttccgagc 20 <210> 478 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 478 ctctaggtcc cggttccgag 20 <210> 479 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 479 cctctaggtc ccggttccga 20 <210> 480 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 480 gcctctaggt cccggttccg 20 <210> 481 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 481 catccgctcc tgcaactgcc 20 <210> 482 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 482 ccatccgctc ctgcaactgc 20 <210> 483 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 483 tccatccgct cctgcaactg 20 <210> 484 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 484 ctccatccgc tcctgcaact 20 <210> 485 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 485 actccatccg ctcctgcaac 20 <210> 486 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 486 aactccatcc gctcctgcaa 20 <210> 487 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 487 caactccatc cgctcctgca 20 <210> 488 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 488 agcaactcca tccgctcctg 20 <210> 489 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 489 cagcaactcc atccgctcct 20 <210> 490 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 490 gcagcaactc catccgctcc 20 <210> 491 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 491 cagctgtggc tccctctgcc 20 <210> 492 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 492 acagctgtgg ctccctctgc 20 <210> 493 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 493 gacagctgtg gctccctctg 20 <210> 494 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 494 tgacagctgt ggctccctct 20 <210> 495 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 495 gtgacagctg tggctccctc 20 <210> 496 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 496 cgtgacagct gtggctccct 20 <210> 497 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 497 ccgtgacagc tgtggctccc 20 <210> 498 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 498 cccgtgacag ctgtggctcc 20 <210> 499 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 499 ccccgtgaca gctgtggctc 20 <210> 500 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 500 cccccgtgac agctgtggct 20 <210> 501 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 501 acccccgtga cagctgtggc 20 <210> 502 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 502 gacccccgtg acagctgtgg 20 <210> 503 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 503 ggacccccgt gacagctgtg 20 <210> 504 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 504 gggacccccg tgacagctgt 20 <210> 505 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 505 gaaggtggat ccgtggcccg 20 <210> 506 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 506 ggaaggtgga tccgtggccc 20 <210> 507 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 507 gggaaggtgg atccgtggcc 20 <210> 508 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 508 tgggaaggtg gatccgtggc 20 <210> 509 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 509 atgggaaggt ggatccgtgg 20 <210> 510 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 510 gatgggaagg tggatccgtg 20 <210> 511 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 511 tagatgggaa ggtggatccg 20 <210> 512 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 512 ctagatggga aggtggatcc 20 <210> 513 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 513 tctagatggg aaggtggatc 20 <210> 514 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 514 atctagatgg gaaggtggat 20 <210> 515 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 515 ccatctagat gggaaggtgg 20 <210> 516 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 516 gccatctaga tgggaaggtg 20 <210> 517 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 517 ggccatctag atgggaaggt 20 <210> 518 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 518 caccagcggg cactggccca 20 <210> 519 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 519 ccaccagcgg gcactggccc 20 <210> 520 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 520 cccaccagcg ggcactggcc 20 <210> 521 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 521 ccccaccagc gggcactggc 20 <210> 522 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 522 ggccccacca gcgggcactg 20 <210> 523 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 523 tggccccacc agcgggcact 20 <210> 524 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 524 ctggccccac cagcgggcac 20 <210> 525 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 525 cctggcccca ccagcgggca 20 <210> 526 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 526 gcctggcccc accagcgggc 20 <210> 527 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 527 gggcctggcc ccaccagcgg 20 <210> 528 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 528 aggtggcggc ggtgcatggg 20 <210> 529 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 529 caggtggcgg cggtgcatgg 20 <210> 530 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 530 gcaggtggcg gcggtgcatg 20 <210> 531 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 531 agcaggtggc ggcggtgcat 20 <210> 532 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 532 cagcaggtgg cggcggtgca 20 <210> 533 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 533 gcagcaggtg gcggcggtgc 20 <210> 534 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 534 agcagcaggt ggcggcggtg 20 <210> 535 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 535 gagcagcagg tggcggcggt 20 <210> 536 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 536 ggagcagcag gtggcggcgg 20 <210> 537 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 537 gggagcagca ggtggcggcg 20 <210> 538 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 538 agggagcagc aggtggcggc 20 <210> 539 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 539 cagggagcag caggtggcgg 20 <210> 540 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 540 gcagggagca gcaggtggcg 20 <210> 541 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 541 ggcagggagc agcaggtggc 20 <210> 542 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 542 tggcagggag cagcaggtgg 20 <210> 543 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 543 ctggcaggga gcagcaggtg 20 <210> 544 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 544 ccctggcagg gagcagcagg 20 <210> 545 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 545 accctggcag ggagcagcag 20 <210> 546 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 546 gaccctggca gggagcagca 20 <210> 547 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 547 ggaccctggc agggagcagc 20 <210> 548 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 548 ggcctaggga ccctggcagg 20 <210> 549 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 549 aggcctaggg accctggcag 20 <210> 550 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 550 ccaggcctag ggaccctggc 20 <210> 551 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 551 gccaggccta gggaccctgg 20 <210> 552 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 552 ggccaggcct agggaccctg 20 <210> 553 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 553 aggccaggcc tagggaccct 20 <210> 554 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 554 taggccaggc ctagggaccc 20 <210> 555 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 555 ataggccagg cctagggacc 20 <210> 556 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 556 gataggccag gcctagggac 20 <210> 557 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 557 cgataggcca ggcctaggga 20 <210> 558 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 558 ccgataggcc aggcctaggg 20 <210> 559 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 559 tccgataggc caggcctagg 20 <210> 560 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 560 ctccgatagg ccaggcctag 20 <210> 561 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 561 cctccgatag gccaggccta 20 <210> 562 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 562 gcctccgata ggccaggcct 20 <210> 563 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 563 gcgcctccga taggccaggc 20 <210> 564 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 564 aacaggagca gggaaagcgc 20 <210> 565 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 565 gaacaggagc agggaaagcg 20 <210> 566 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 566 cgaacaggag cagggaaagc 20 <210> 567 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 567 gcgaacagga gcagggaaag 20 <210> 568 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 568 ggcgaacagg agcagggaaa 20 <210> 569 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 569 cggcgaacag gagcagggaa 20 <210> 570 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 570 acggcgaaca ggagcaggga 20 <210> 571 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 571 aacggcgaac aggagcaggg 20 <210> 572 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 572 caacggcgaa caggagcagg 20 <210> 573 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 573 gggcggcggc acgagacaga 20 <210> 574 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 574 agggcggcgg cacgagacag 20 <210> 575 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 575 cagggcggcg gcacgagaca 20 <210> 576 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 576 ccagggcggc ggcacgagac 20 <210> 577 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 577 cccagggcgg cggcacgaga 20 <210> 578 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 578 gcccagggcg gcggcacgag 20 <210> 579 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 579 agcccagggc ggcggcacga 20 <210> 580 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 580 cagcccaggg cggcggcacg 20 <210> 581 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 581 gcagcccagg gcggcggcac 20 <210> 582 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 582 ctgcggtgag ttggccggcg 20 <210> 583 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 583 actgcggtga gttggccggc 20 <210> 584 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 584 gactgcggtg agttggccgg 20 <210> 585 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 585 agactgcggt gagttggccg 20 <210> 586 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 586 cagactgcgg tgagttggcc 20 <210> 587 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 587 ccagactgcg gtgagttggc 20 <210> 588 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 588 gccagactgc ggtgagttgg 20 <210> 589 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 589 cgccagactg cggtgagttg 20 <210> 590 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 590 aagacagttc tagggttcag 20 <210> 591 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 591 gaagacagtt ctagggttca 20 <210> 592 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 592 cgaagacagt tctagggttc 20 <210> 593 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 593 tcgaagacag ttctagggtt 20 <210> 594 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 594 gtcgaagaca gttctagggt 20 <210> 595 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 595 agtcgaagac agttctaggg 20 <210> 596 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 596 gagtcgaaga cagttctagg 20 <210> 597 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 597 ggagtcgaag acagttctag 20 <210> 598 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 598 cggagtcgaa gacagttcta 20 <210> 599 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 599 ccggagtcga agacagttct 20 <210> 600 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 600 cccggagtcg aagacagttc 20 <210> 601 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 601 ccccggagtc gaagacagtt 20 <210> 602 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 602 gccccggagt cgaagacagt 20 <210> 603 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 603 ggccccggag tcgaagacag 20 <210> 604 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 604 gggccccgga gtcgaagaca 20 <210> 605 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 605 aggcggtggg cgcggcttct 20 <210> 606 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 606 caggcggtgg gcgcggcttc 20 <210> 607 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 607 gcaggcggtg ggcgcggctt 20 <210> 608 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 608 tggcaggcgg tgggcgcggc 20 <210> 609 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 609 actggcaggc ggtgggcgcg 20 <210> 610 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 610 gaactggcag gcggtgggcg 20 <210> 611 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 611 tgaactggca ggcggtgggc 20 <210> 612 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 612 tgtgaactgg caggcggtgg 20 <210> 613 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 613 tggagctggg cggagaccca 20 <210> 614 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 614 actggagctg ggcggagacc 20 <210> 615 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 615 gactggagct gggcggagac 20 <210> 616 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 616 aggactggag ctgggcggag 20 <210> 617 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 617 acaggactgg agctgggcgg 20 <210> 618 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 618 cacaggactg gagctgggcg 20 <210> 619 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 619 tcacaggact ggagctgggc 20 <210> 620 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 620 gcctcagcct ggccgaaaga 20 <210> 621 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 621 ggcctcagcc tggccgaaag 20 <210> 622 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 622 tggtggagcc aagccctccc 20 <210> 623 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 623 gggcaccctc agagcctgaa 20 <210> 624 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 624 accccactgc aagaagtcgg 20 <210> 625 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 625 gccccaggat gggaggatct 20 <210> 626 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 626 cataggacag agaaatgttg 20 <210> 627 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 627 tgctgacctt actctgcccc 20 <210> 628 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 628 taagccatgg ctctgagtca 20 <210> 629 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 629 agagaggcca tgggaggctg 20 <210> 630 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 630 ctggccctcc tggcttgccc 20 <210> 631 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 631 agctgcccca tgctggccct 20 <210> 632 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 632 gcccctggca gctgccccat 20 <210> 633 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 633 ctgtcggctg cgcccctggc 20 <210> 634 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 634 cgccgaacac ctgcctgtcg 20 <210> 635 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 635 cctcccagtg cctgggcacc 20 <210> 636 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 636 gcgcctgtct gcaaagctgg 20 <210> 637 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 637 cccaaagttg tccctcctgg 20 <210> 638 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 638 acacccagaa gaacccaaag 20 <210> 639 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 639 ctgacccaca cggctcatag 20 <210> 640 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 640 tggccccagg ccctggaaag 20 <210> 641 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 641 gacaaggcag ctggcagaag 20 <210> 642 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 642 aagaaaccag tgaccagtga 20 <210> 643 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 643 ctgtgaaatg ggaggaggag 20 <210> 644 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 644 gaaggttttt ccagaggctg 20 <210> 645 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 645 ggccaggaga gtcattaggg 20 <210> 646 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 646 ccacaaaagg agtgctcctc 20 <210> 647 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 647 ccttttaagg cagcaggaac 20 <210> 648 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 648 ctaggactgt ctgcttccca 20 <210> 649 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 649 gtcattcatc aatttctaag 20 <210> 650 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 650 ggaggagctg cagccggaga 20 <210> 651 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 651 gcacccggag gagctgcagc 20 <210> 652 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 652 gcacgacacc tgcagggcac 20 <210> 653 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 653 agctcaccag gtagttctca 20 <210> 654 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 654 gcttcctctc cccacctcct 20 <210> 655 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 655 gcagcacccc caatcctaga 20 <210> 656 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 656 gcccctcatc cacctgacac 20 <210> 657 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 657 ttccaggtaa gagacccccc 20 <210> 658 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 658 agaataggtc ccagacactc 20 <210> 659 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 659 ctccccctga gatgttctgg 20 <210> 660 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 660 ccccagccca gagataacca 20 <210> 661 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 661 cctgatccat cacggatggc 20 <210> 662 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 662 tactccatga ccaggtactg 20 <210> 663 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 663 gctctgacct tccaagaacc 20 <210> 664 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 664 ctcccttctg tggtcccacc 20 <210> 665 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 665 gtcgggtttg atgtccctgc 20 <210> 666 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 666 agggcactgg ctcaccgttc 20 <210> 667 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 667 gggccctcct tccaaccact 20 <210> 668 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 668 gcccacccct ctgggcccac 20 <210> 669 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 669 aggagcagag cgaggcttgg 20 <210> 670 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 670 caccttgtag tggacgatct 20 <210> 671 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 671 ctaccccgcc cccgctcacc 20 <210> 672 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 672 ctaggtcact gctgggtcct 20 <210> 673 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 673 ctcagatagc tccccactcc 20 <210> 674 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 674 aattctctaa ttctctagac 20 <210> 675 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 675 tacctgaggg ccatgcagga 20 <210> 676 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 676 gttccaagac tgatcctgca 20 <210> 677 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 677 aggagggcgg tggcgcggcg 20 <210> 678 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 678 tgacagctgg aaggagaaga 20 <210> 679 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 679 catgggaagg tggatccgtg 20 <210> 680 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 680 ggaggttatc tagggagatc 20 <210> 681 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 681 gaagggacag gtgacccgat 20 <210> 682 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 682 cgtaccctgg cagggagcag 20 <210> 683 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 683 ggactcgccc cgcctacgcc 20 <210> 684 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 684 ctcctgggac tcgccccgcc 20 <210> 685 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 685 gctcctggga ctcgccccgc 20 <210> 686 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 686 attggctcct gggactcgcc 20 <210> 687 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 687 gattggctcc tgggactcgc 20 <210> 688 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 688 gcctctgatt ggctcctggg 20 <210> 689 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 689 gcatgggcct ctgattggct 20 <210> 690 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 690 cacccggcat gggcctctga 20 <210> 691 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 691 gccaggccta gggacctgcg 20 <210> 692 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 692 ttcctccccc aaccctgatt 20 <210> 693 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 693 aagtttgcag caacttttct 20 <210> 694 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 694 gcccctcgga attcccggct 20 <210> 695 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 695 catctcggcc tgcgctccgc 20 <210> 696 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 696 gcaggccccc acattcccca 20 <210> 697 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 697 cttctgcacg cctccgtctc 20 <210> 698 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 698 tggcccacag ccacggccgg 20 <210> 699 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 699 ggcctggccc caccagcggg 20 <210> 700 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 700 cctggcaggg agcagcaggt 20 <210> 701 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 701 cagccgcact tcggctgaca 20 <210> 702 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 702 gcctgggtcc agcaccagct 20 <210> 703 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 703 gtcccaggaa gcctgggtcc 20 <210> 704 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 704 cgttagcagg tccccgccca 20 <210> 705 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 705 gtctatggcc atgacaatct 20 <210> 706 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 706 gtagcccagc cggtgcacgg 20 <210> 707 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 707 gggtgcccac agccaccagc 20 <210> 708 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 708 tggcccgtag ctgcctgccc 20 <210> 709 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 709 ggaaatcacc tgccccacct 20 <210> 710 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 710 ggatgtttct ggaaatcacc 20 <210> 711 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 711 gtggcaccct cgaagtctgg 20 <210> 712 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 712 ccccgctcac catggcagtg 20 <210> 713 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 713 ggtccgggac ctgattgtct 20 <210> 714 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 714 gctgcatgtc tgcccgtccc 20 <210> 715 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 715 ggccccagaa ccctagctgc 20 <210> 716 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 716 tcacagggcc tggctgcccc 20 <210> 717 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 717 ggctgacatg ttgggcaggc 20 <210> 718 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 718 tgtccaggcc ccagaaccct 20 <210> 719 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 719 ggccaggcct agggatctgc 20 <210> 720 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 720 cgcctcggat aggccaggcc 20 <210> 721 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 721 ggcttggagt cttagggttc 20 <210> 722 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 722 tccccggccg ccaggtggca 20 <210> 723 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 723 ggtgctgggc acgagccctg 20 <210> 724 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 724 gcccagctgc tgcagcagcg 20 <210> 725 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 725 ccgtgtgtgc tggcagaggt 20 <210> 726 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 726 ataaataccg aggaatgtcg 20 <210> 727 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 727 gggacagaca ataaataccg 20 <210> 728 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 728 gtgcagccca gtgtggcggc 20 <210> 729 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 729 cctggagaag ttctggttgg 20 <210> 730 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 730 ggtgacccga tcggagccca 20 <210> 731 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 731 agctggagag agaagggaca 20 <210> 732 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 732 gtgagggact cgcctgcggc 20 <210> 733 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 733 gcggctgcgg tgccccagcc 20 <210> 734 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 734 gggccatcta gctggagaga 20 <210> 735 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 735 ccccactgca agaagtcggc 20 <210> 736 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 736 ttgagccctt ttaaggcagc 20 <210> 737 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 737 tgaccaggta ctgggagcgg 20 <210> 738 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 738 cctggagctg gatcagtccc 20 <210> 739 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 739 acatgggaag gtggatccgt 20 <210> 740 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 740 gtgggacata ccctggcagg 20 <210> 741 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 741 gccaggccta gggatctgca 20 <210> 742 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 742 ggaagcacga cacctcgcct 20 <210> 743 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 743 cctcaccatt ccatcaggct 20 <210> 744 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 744 cggcagcgac aagtgttccc 20 <210> 745 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 745 gtctctgaag gccatgcagc 20 <210> 746 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 746 cagccacttg atccggtggg 20 <210> 747 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 747 aggtcggcct cttcagccac 20 <210> 748 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 748 gttggctgga gaagttctgg 20 <210> 749 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 749 ccccgtgatg gctgcggctc 20 <210> 750 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 750 aggccaggcc tagggatcct 20 <210> 751 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 751 ggcgcggtgc cccagcctgg 20 <210> 752 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 752 gtcctggccc caccagcggg 20 <210> 753 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 753 ccaggcctag gaatcctggc 20 <210> 754 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 754 gcgcctcgga tagccaggcc 20 <210> 755 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 755 cccagtgtgg cgcagcagcc 20 <210> 756 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 756 gtgtttcatc ttcaccaccg 20 <210> 757 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 757 aggtcagcct cttcagccac 20 <210> 758 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 758 ggccatatgg gaaggtggat 20 <210> 759 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 759 ggaggatttg gcgagaagca 20 <210> 760 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 760 cgaagtctgc cccacctcga 20 <210> 761 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 761 gtggcaccct cgaagtctgc 20 <210> 762 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 762 gggtccattg taaggaagct 20 <210> 763 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 763 ggtgcccaca gccaccaggg 20 <210> 764 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 764 tccatggcag tgagccggtc 20 <210> 765 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 765 gggaccactt gatccggtgg 20 <210> 766 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 766 ggatcagagt tgggaccact 20 <210> 767 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 767 ccccgtgatg gctgcggttc 20 <210> 768 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 768 gtgtgtcctc ataccccgcc 20 <210> 769 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 769 gcaccctcga agtctcgacc 20 <210> 770 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 770 gctctgaagg ccatgcagca 20 <210> 771 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 771 gacatatgcc aagattgtgc actac 25 <210> 772 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 772 cacgaatgag gtcctgagct t 21 <210> 773 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 773 aacacttgtc gctgccgctg gc 22 <210> 774 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 774 agcgaggctt cacttggcgc 20 <210> 775 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 775 gggaagcgag gcttcacttg 20 <210> 776 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 776 gcggtcagcg atcccagggt 20 <210> 777 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 777 gggtgccagc gcggtgatct 20 <210> 778 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 778 tgttacaaag aaagtgactg 20 <210> 779 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 779 cgatggcagc aacggaagtt 20 <210> 780 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 780 gtcagtttac gatggcagca 20 <210> 781 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 781 cagggctttg tttcgaaaaa 20 <210> 782 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 782 ccattttctt ccacagggct 20 <210> 783 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 783 atgcttcttc aagttttcca 20 <210> 784 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 784 cagaatgact ttaatgcttc 20 <210> 785 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 785 ccaccgcaaa tgcttctaga c 21 <210> 786 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 786 cccccccatt gagaagattc 20 <210> 787 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 787 ctccacctcc agcacgcgac ttct 24 <210> 788 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 788 gcggtcagcg atcccagggt 20 <210> 789 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 789 agcagcagca gcagcagcag cagca 25 <210> 790 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 790 agcagcagca gcagcagcag 20 <210> 791 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 791 gcagcagcag cagca 15 <210> 792 <400> 792 000 <210> 793 <211> 2611 <212> DNA <213> Mus musculus <400> 793 cgggaagacc ccgagctccg gcccggggag gggccatggt gttgcctgcc caacatgtca 60 gccgaagtgc ggctgaggca gctccagcag ctggtgctgg acccaggctt cctgggactg 120 gagcccctgc tcgaccttct cctgggcgtc caccaggagc tgggtgcctc tcacctagcc 180 caggacaagt atgtggccga cttcttgcag tgggtggagc ccattgcagc aaggcttaag 240 gaggtccgac tgcagaggga tgattttgag attttgaagg tgatcgggcg tggggcgttc 300 agcgaggtag cggtggtgaa gatgaaacag acgggccaag tgtatgccat gaagattatg 360 aataagtggg acatgctgaa gagaggcgag gtgtcgtgct tccgggaaga aagggatgta 420 ttagtgaaag gggaccggcg ctggatcaca cagctgcact ttgccttcca ggatgagaac 480 tacctgtacc tggtcatgga atactacgtg ggcggggacc tgctaacgct gctgagcaag 540 tttggggagc ggatccccgc cgagatggct cgcttctacc tggccgagat tgtcatggcc 600 atagactccg tgcaccggct gggctacgtg cacagggaca tcaaaccaga taacattctg 660 ctggaccgat gtgggcacat tcgcctggca gacttcggct cctgcctcaa actgcagcct 720 gatggaatgg tgaggtcgct ggtggctgtg ggcaccccgg actacctgtc tcctgagatt 780 ctgcaggccg ttggtggagg gcctggggca ggcagctacg ggccagagtg tgactggtgg 840 gcactgggcg tgttcaccta tgagatgttc tatgggcaga cccccttcta cgcggactcc 900 acagccgaga catatgccaa gattgtgcac tacagggaac acttgtcgct gccgctggca 960 gacacagttg tccccgagga agctcaggac ctcattcgtg ggctgctgtg tcctgctgag 1020 ataaggctag gtcgaggtgg ggcagacttc gagggtgcca cggacacatg caatttcgat 1080 gtggtggagg accggctcac tgccatggtg agcgggggcg gggagacgct gtcagacatg 1140 caggaagaca tgccccttgg ggtgcgcctg cccttcgtgg gctactccta ctgctgcatg 1200 gccttcagag acaatcaggt cccggacccc acccctatgg aactagaggc cctgcagttg 1260 cctgtgtcag acttgcaagg gcttgacttg cagcccccag tgtccccacc ggatcaagtg 1320 gctgaagagg ctgacctagt ggctgtccct gcccctgtgg ctgaggcaga gaccacggta 1380 acgctgcagc agctccagga agccctggaa gaagaggttc tcacccggca gagcctgagc 1440 cgcgagctgg aggccatccg gaccgccaac cagaacttct ccagccaact acaggaggcc 1500 gaggtccgaa accgagacct ggaggcgcat gttcggcagc tacaggaacg gatggagatg 1560 ctgcaggccc caggagccgc agccatcacg ggggtcccca gtccccgggc cacggatcca 1620 ccttcccatc tagatggccc cccggccgtg gctgtgggcc agtgcccgct ggtggggcca 1680 ggccccatgc accgccgtca cctgctgctc cctgccagga tccctaggcc tggcctatcc 1740 gaggcgcgtt gcctgctcct gttcgccgct gctctggctg ctgccgccac actgggctgc 1800 actgggttgg tggcctatac cggcggtctc accccagtct ggtgtttccc gggagccacc 1860 ttcgccccct gaaccctaag actccaagcc atctttcatt taggcctcct aggaaggtcg 1920 agcgaccagg gagcgaccca aagcgtctct gtgcccatcg cccccccccc cccccccacc 1980 gctccgctcc acacttctgt gagcctgggt ccccacccag ctccgctcct gtgatccagg 2040 cctgccacct ggcggccggg gagggaggaa cagggctcgt gcccagcacc cctggttcct 2100 gcagagctgg tagccaccgc tgctgcagca gctgggcatt cgccgacctt gctttactca 2160 gccccgacgt ggatgggcaa actgctcagc tcatccgatt tcactttttc actctcccag 2220 ccatcagtta caagccataa gcatgagccc cctatttcca gggacatccc attcccatag 2280 tgatggatca gcaagacctc tgccagcaca cacggagtct ttggcttcgg acagcctcac 2340 tcctgggggt tgctgcaact ccttccccgt gtacacgtct gcactctaac aacggagcca 2400 cagctgcact cccccctccc ccaaagcagt gtgggtattt attgatcttg ttatctgact 2460 cactgacaga ctccgggacc cacgttttag atgcattgag actcgacatt cctcggtatt 2520 tattgtctgt ccccacctac gacctccact cccgaccctt gcgaataaaa tacttctggt 2580 ctgccctaaa aaaaaaaaaa aaaaaaaaaa a 2611 <210> 794 <211> 988 <212> DNA <213> Mus musculus <220> <221> misc_feature <222> 531, 942 <223> n = A,T,C or G <400> 794 gctggaccgg tccggaattc tccggatcgc cagcctttgt gggccatatt cgtcatccct 60 cctggcttct catctgcttt tgtggtccta gctcaagacc tctaattcct ctgctgactt 120 aaatgccctt ccccagaggt cttctcaggc ctagtggaca agcttggagc cttatctgct 180 cctgcccaac attgagccaa agctccagct taccccagct tccttacaat ggaccccatt 240 gcagcaaggc ttaaggaggt ccgactgcag agggatgatt ttgagatttt gaaggtgatc 300 gggcgtgggg cgttcagcga ggtagcggtg gtgaagatga aacagacggg ccaagtgtat 360 gccatgaaga ttatgaataa gtgggacatg ctgaagagag gcgaggtgtc gtgcttccgg 420 gaagaaaggg atgtattagt gaaaggggac cggcgctgga tcacacagct gcactttgcc 480 ttccaggatg agaactacct gtacctggtc atggaatact acgtgggcgg ngacctgcta 540 acgctgctga gcaagttttg gggagcggat ccccgccgag atggctcgct tctacctggc 600 cgagattgtc atggccatag actccgtgca ccggctgggc tacgtgcaca gggacatcaa 660 accagataac attctgctgg accgatgtgg gcacattcgc ctggcagact tcggctcctg 720 gcctcaactg cagcctgatg gaatggtgga gtcccctggt ggctgtgggc acccccggac 780 tacctgtctc ctgaaattct gcagggcctt ggtgggaggc cctggggaag gcaactacgg 840 gccaaaagtt ggaagggggg ggcctggggg gggttcccct atgaaaagtt ctatggggag 900 gacccccttt aagcggaatc ccaggccgaa aaatatgccc angattgggc cctaacaggg 960 aaaacttttc ccctgcccct gggacaat 988 <210> 795 <211> 649 <212> DNA <213> Mus musculus <400> 795 ggcgtgttcg cctatgagat gttctatggg cagaccccct tctacgcgga ctccacagcc 60 gagacatatg ccaagattgt gcactacagg gaacacttgt cgctgccgct ggcagacaca 120 gttgtccccg aggaagctca ggacctcatt cgtgggctgc tgtgtcctgc tgagataagg 180 ctaggtcgag gtggggcagg tgatttccag aaacatcctt tcttctttgg ccttgattgg 240 gagggtctcc gagacagtgt accccccttt acaccagact tcgagggtgc cacggacaca 300 tgcaatttcg atgtggtgga ggaccggctc actgccatgg agacgctgtc agacatgcag 360 gaagacatgc cccttggggt gcgcctgccc ttcgtgggct actcctactg ctgcatggcc 420 ttcagagaca atcaggtccc ggaccccacc cctatggaac tagaggccct gcagttgcct 480 gtgtcagact tgcaagggct tgacttgcag cccccagtgt ccccaccgga tcaagtggtc 540 ccaactctga tccccaccga caggctgaag aggctgacct agtggctgtc cctgcccctg 600 tggctgaggc agagccacgg taacgctgca gcagctccag gaagccctg 649 <210> 796 <211> 527 <212> DNA <213> Mus musculus <400> 796 atttcgatgt ggtggaggac cggctcactg ccatggtgag cgggggcggg gagacgctgt 60 cagacatgca ggaagacatg ccccttgggg tgcgcctgcc cttcgtgggc tactcctact 120 gctgcatggc cttcagagac aatcaggtcc cggaccccac ccctatggaa ctagaggccc 180 tgcagttgcc tgtgtcagac ttgcaagggc ttgacttgca gcccccagtg tccccaccgg 240 atcaagtggc tgaagaggct gacctagtgg ctgtccctgc ccctgtggct gaggcagaga 300 ccacggtaac gctgcagcag ctccaggaag ccctggaaga agaggttctc acccggcaga 360 gcctgagccg cgagctggag gccatccgga ccgccaacca gaacttctcc aggaggccga 420 ggtccgaaac cgagacctgg aggcgcatgt tcggcagcta caggaacgga tggagatgct 480 gcaggcccca ggaaccgcag ccatcacggg ggtccccagt cccccgg 527 <210> 797 <211> 567 <212> DNA <213> Mus musculus <400> 797 atggtgaggt cgctggtggc tgtgggcacc ccggactacc tgtctcctga gattctgcag 60 gccgttggtg gagggcctgg ggcaggcagc tacgggccag agtgtgactg gtgggcactg 120 ggcgtgttcg cctatgagat gttctatggg cagaccccct tctacgcgga ctccacagcc 180 gagacatatg ccaagattgt gcactacagg gaacacttgt cgctgccgct ggcagacaca 240 gttgtccccg aggaagctca ggacctcatt cgtgggctgc tgtgtcctgc tgagataagg 300 ctaggtcgag gtggggcagg tgatttccag aaacatcctt tcttctttgg ccttgattgg 360 gagggtctcc gagacagtgt accccccttt acaccagact tcgagggtgc cacggacaca 420 tgcaatttcg atgtggtgga ggaccggctc actgccatgg tgagcggggg cggggtatga 480 ggacacacag gtgaccagtc cccaagacag tgagtgaggc ttcactcttg gcagtactaa 540 aattgaatgt agggggctgg gctcttg 567 <210> 798 <211> 2474 <212> DNA <213> Mus musculus <400> 798 ccgggaagaa agggatgtat tagtgaaagg ggaccggcgc tggatcacac agctgcactt 60 tgccttccag gatgagaact acctgtacct ggtcatggaa tactacgtgg gcggggacct 120 gctaacgctg ctgagcaagt ttggggagcg gatccccgcc gagatggctc gcttctacct 180 ggccgagatt gtcatggcca tagactccgt gcaccggctg ggctacgtgc acagggacat 240 caaaccagat aacattctgc tggaccgatg tgggcacatt cgcctggcag acttcggctc 300 ctgcctcaaa ctgcagcctg atggaatggt gaggtcgctg gtggctgtgg gcaccccgga 360 ctacctgtct cctgagattc tgcaggccgt tggtggaggg cctggggcag gcagctacgg 420 gccagagtgt gactggtggg cactgggcgt gttcgcctat gagatgttct atgggcagac 480 ccccttctac gcggactcca cagccgagac atatgccaag attgtgcact acagggaaca 540 cttgtcgctg ccgctggcag acacagttgt ccccgaggaa gctcaggacc tcattcgtgg 600 gctgctgtgt cctgctgaga taaggctagg tcgagacttc gagggtgcca cggacacatg 660 caatttcgat gtggtggagg accggctcac tgccatggtg agcgggggcg gggagacgct 720 gtcagacatg caggaagaca tgccccttgg ggtgcgcctg cccttcgtgg gctactccta 780 ctgctgcatg gccttcagag acaatcaggt cccggacccc acccctatgg aactagaggc 840 cctgcagttg cctgtgtcag acttgcaagg gcttgacttg cagcccccag tgtccccacc 900 ggatcaagtg gctgaagagg ccgacctagt ggctgtccct gcccctgtgg ctgaggcaga 960 gaccacggta acgctgcagc agctccagga agccctggaa gaagaggttc tcacccggca 1020 gagcctgagc cgcgagctgg aggccatccg gaccgccaac cagaacttct ccagccaact 1080 acaggaggcc gaggtccgaa accgagacct ggaggcgcat gttcggcagc tacaggaacg 1140 gatggagatg ctgcaggccc caggagccgc aggcgagtcc ctcacctgct tccagccaag 1200 ggggcactgg gtggagatgg ggggcatgtt gggtgtgtga accctcgggg caggggagga 1260 gtccaggctg gggcaccgca gccgcgccac tgcctttctc ctccatcctc cacactccat 1320 acacctctct cttctccttc cagccatcac gggggtcccc agtccccggg ccacggatcc 1380 accttcccat gcttctcgcc aaatcctccc caagggaact ccctagactc ccgttctggc 1440 ctcgactaga ttcccgcact gcctctcgcc ctgctgctgg gctccgatcg ggtcacctgt 1500 cccttctctc tccagctaga tggccccccg gccgtggctg tgggccagtg cccgctggtg 1560 gggccaggcc ccatgcaccg ccgtcacctg ctgctccctg ccaggatccc taggcctggc 1620 ctatccgagg cgcgttgcct gctcctgttc gccgctgctc tggctgctgc cgccacactg 1680 ggctgcactg ggttggtggc ctataccggc ggtctcaccc cagtctggtg tttcccggga 1740 gccaccttcg ccccctgaac cctaagactc caagccatct ttcatttagg cctcctagga 1800 agatcgagcg accagggagc gacccaaagc gtctctgtgc ccatcgcccc cccccccccc 1860 cccaccgctc cgctccacac ttctgtgagc ctgggtcccc acccagctcc gctcctgtga 1920 tccaggcctg ccacctggcg gccggggagg gaggaacagg gctcgtgccc agcacccctg 1980 gttcctgcag agctggtagc caccgctgct gcagcagctg ggcattcgcc gaccttgctt 2040 tactcagccc tgacgtggat gggctaactg ctcagctcat ccgatttcac tttttcactc 2100 tcccagccat cagttacaag ccataagcat gagcccccta tttccaggga catcccattc 2160 ccatagtgat ggatcagcaa gacctctgcc agcacacacg gagtctttgg cttcggacag 2220 cctcactcct gggggttgct gcaactcctt ccccgtgtac acgtctgcac tctaacaacg 2280 gagccacagc tgcactcccc cctcccccaa agcagtgtgg gtatttattg atcttgttat 2340 ctgactcact gacagactcc gggacccacg ttttagatgc attgagactc gacattcctc 2400 ggtatttatt gtctgtcccc acctacgacc tccactcccg acccttgcga ataaaatact 2460 tctggtctgc ccta 2474 <210> 799 <211> 2135 <212> DNA <213> Mus musculus <400> 799 ccgggaagaa agggatgtat tagtgaaagg ggaccggcgc tggatcacac agctgcactt 60 tgccttccag gatgagaact acctgtacct ggtcatggaa tactacgtgg gcggggacct 120 gctaacgctg ctgagcaagt ttggggagcg gatccccgcc gagatggctc gcttctacct 180 ggccgagatt gtcatggcca tagactccgt gcaccggctg ggctacgtgc acagggacat 240 caaaccagat aacattctgc tggaccgatg tgggcacatt cgcctggcag acttcggctc 300 ctgcctcaaa ctgcagcctg atggaatggt gaggtcgctg gtggctgtgg gcaccccgga 360 ctacctgtct cctgagattc tgcaggccgt tggtggaggg cctggggcag gcagctacgg 420 gccagagtgt gactggtggg cactgggcgt gttcgcctat gagatgttct atgggcagac 480 ccccttctac gcggactcca cagccgagac atatgccaag attgtgcact acagggaaca 540 cttgtcgctg ccgctggcag acacagttgt ccccgaggaa gctcaggacc tcattcgtgg 600 gctgctgtgt cctgctgaga taaggctagg tcgaggtggg gcaggtgatt tccagaaaca 660 tcctttcttc tttggccttg attgggaggg tctccgagac agtgtacccc cctttacacc 720 agacttcgag ggtgccacgg acacatgcaa tttcgatgtg gtggaggacc ggctcactgc 780 catggagacg ctgtcagaca tgcaggaaga catgcccctt ggggtgcgcc tgcccttcgt 840 gggctactcc tactgctgca tggccttcag agctgaagag gccgacctag tggctgtccc 900 tgcccctgtg gctgaggcag agaccacggt aacgctgcag cagctccagg aagccctgga 960 agaagaggtt ctcacccggc agagcctgag ccgcgagctg gaggccatcc ggaccgccaa 1020 ccagaacttc tccagccaac tacaggaggc cgaggtccga aaccgagacc tggaggcgca 1080 tgttcggcag ctacaggaac ggatggagat gctgcaggcc ccaggagccg cagccatcac 1140 gggggtcccc agtccccggg ccacggatcc accttcccat atggcccccc ggccgtggct 1200 gtgggccagt gcccgctggt ggggccaggc cccatgcacc gccgtcacct gctgctccct 1260 gccaggatcc ctaggcctgg cctatccgag gcgcgttgcc tgctcctgtt cgccgctgct 1320 ctggctgctg ccgccacact gggctgcact gggttggtgg cctataccgg cggtctcacc 1380 ccagtctggt gtttcccggg agccaccttc gccccctgaa ccctaagact ccaagccatc 1440 tttcatttag gcctcctagg aagatcgagc gaccagggag cgacccaaag cgtctctgtg 1500 cccatcgccc cccccccccc ccccaccgct ccgctccaca cttctgtgag cctgggtccc 1560 cacccagctc cgctcctgtg atccaggcct gccacctggc ggccggggag ggaggaacag 1620 ggctcgtgcc cagcacccct ggttcctgca gagctggtag ccaccgctgc tgcagcagct 1680 gggcattcgc cgaccttgct ttactcagcc ctgacgtgga tgggctaact gctcagctca 1740 tccgatttca ctttttcact ctcccagcca tcagttacaa gccataagca tgagccccct 1800 atttccaggg acatcccatt cccatagtga tggatcagca agacctctgc cagcacacac 1860 ggagtctttg gcttcggaca gcctcactcc tgggggttgc tgcaactcct tccccgtgta 1920 cacgtctgca ctctaacaac ggagccacag ctgcactccc ccctccccca aagcagtgtg 1980 ggtatttatt gatcttgtta tctgactcac tgacagactc cgggacccac gttttagatg 2040 cattgagact cgacattcct cggtatttat tgtctgtccc cacctacgac ctccactccc 2100 gacccttgcg aataaaatac ttctggtctg cccta 2135 <210> 800 <211> 2873 <212> DNA <213> Homo sapiens <400> 800 aggggggctg gaccaagggg tggggagaag gggaggaggc ctcggccggc cgcagagaga 60 agtggccaga gaggcccagg ggacagccag ggacaggcag acatgcagcc agggctccag 120 ggcctggaca ggggctgcca ggccctgtga caggaggacc ccgagccccc ggcccgggga 180 ggggccatgg tgctgcctgt ccaacatgtc agccgaggtg cggctgaggc ggctccagca 240 gctggtgttg gacccgggct tcctggggct ggagcccctg ctcgaccttc tcctgggcgt 300 ccaccaggag ctgggcgcct ccgaactggc ccaggacaag tacgtggccg acttcttgca 360 gtgggcggag cccatcgtgg tgaggcttaa ggaggtccga ctgcagaggg acgacttcga 420 gattctgaag gtgatcggac gcggggcgtt cagcgaggta gcggtagtga agatgaagca 480 gacgggccag gtgtatgcca tgaagatcat gaacaagtgg gacatgctga agaggggcga 540 ggtgtcgtgc ttccgtgagg agagggacgt gttggtgaat ggggaccggc ggtggatcac 600 gcagctgcac ttcgccttcc aggatgagaa ctacctgtac ctggtcatgg agtattacgt 660 gggcggggac ctgctgacac tgctgagcaa gtttggggag cggattccgg ccgagatggc 720 gcgcttctac ctggcggaga ttgtcatggc catagactcg gtgcaccggc ttggctacgt 780 gcacagggac atcaaacccg acaacatcct gctggaccgc tgtggccaca tccgcctggc 840 cgacttcggc tcttgcctca agctgcgggc agatggaacg gtgcggtcgc tggtggctgt 900 gggcacccca gactacctgt cccccgagat cctgcaggct gtgggcggtg ggcctgggac 960 aggcagctac gggcccgagt gtgactggtg ggcgctgggt gtattcgcct atgaaatgtt 1020 ctatgggcag acgcccttct acgcggattc cacggcggag acctatggca agatcgtcca 1080 ctacaaggag cacctctctc tgccgctggt ggacgaaggg gtccctgagg aggctcgaga 1140 cttcattcag cggttgctgt gtcccccgga gacacggctg ggccggggtg gagcaggcga 1200 cttccggaca catcccttct tctttggcct cgactgggat ggtctccggg acagcgtgcc 1260 cccctttaca ccggatttcg aaggtgccac cgacacatgc aacttcgact tggtggagga 1320 cgggctcact gccatggaga cactgtcgga cattcgggaa ggtgcgccgc taggggtcca 1380 cctgcctttt gtgggctact cctactcctg catggccctc agggacagtg aggtcccagg 1440 ccccacaccc atggaactgg aggccgagca gctgcttgag ccacacgtgc aagcgcccag 1500 cctggagccc tcggtgtccc cacaggatga aacagctgaa gtggcagttc cagcggctgt 1560 ccctgcggca gaggctgagg ccgaggtgac gctgcgggag ctccaggaag ccctggagga 1620 ggaggtgctc acccggcaga gcctgagccg ggagatggag gccatccgca cggacaacca 1680 gaacttcgcc agtcaactac gcgaggcaga ggctcggaac cgggacctag aggcacacgt 1740 ccggcagttg caggagcgga tggagttgct gcaggcagag ggagccacag ctgtcacggg 1800 ggtccccagt ccccgggcca cggatccacc ttcccatatg gccccccggc cgtggctgtg 1860 ggccagtgcc cgctggtggg gccaggcccc atgcaccgcc gccacctgct gctccctgcc 1920 agggtcccta ggcctggcct atcggaggcg ctttccctgc tcctgttcgc cgttgttctg 1980 tctcgtgccg ccgccctggg ctgcattggg ttggtggccc acgccggcca actcaccgca 2040 gtctggcgcc gcccaggagc cgcccgcgct ccctgaaccc tagaactgtc ttcgactccg 2100 gggccccgtt ggaagactga gtgcccgggg cacggcacag aagccgcgcc caccgcctgc 2160 cagttcacaa ccgctccgag cgtgggtctc cgcccagctc cagtcctgtg atccgggccc 2220 gccccctagc ggccggggag ggaggggccg ggtccgcggc cggcgaacgg ggctcgaagg 2280 gtccttgtag ccgggaatgc tgctgctgct gctgctgctg ctgctgctgc tgctgctgct 2340 gctgctgctg ctgctgctgg ggggatcaca gaccatttct ttctttcggc caggctgagg 2400 ccctgacgtg gatgggcaaa ctgcaggcct gggaaggcag caagccgggc cgtccgtgtt 2460 ccatcctcca cgcaccccca cctatcgttg gttcgcaaag tgcaaagctt tcttgtgcat 2520 gacgccctgc tctggggagc gtctggcgcg atctctgcct gcttactcgg gaaatttgct 2580 tttgccaaac ccgctttttc ggggatcccg cgcccccctc ctcacttgcg ctgctctcgg 2640 agccccagcc ggctccgccc gcttcggcgg tttggatatt tattgacctc gtcctccgac 2700 tcgctgacag gctacaggac ccccaacaac cccaatccac gttttggatg cactgagacc 2760 ccgacattcc tcggtattta ttgtctgtcc ccacctagga cccccacccc cgaccctcgc 2820 gaataaaagg ccctccatct gcccaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 2873 <210> 801 <211> 1509 <212> DNA <213> Homo sapiens <400> 801 ccaccgcagc ggacagcgcc aagtgaagcc tcgcttcccc tccgcggcga ccagggcccg 60 agccgagagt agcagttgta gctacccgcc cagaaactag acacaatgtg cgacgaagac 120 gagaccaccg ccctcgtgtg cgacaatggc tccggcctgg tgaaagccgg cttcgccggg 180 gatgacgccc ctagggccgt gttcccgtcc atcgtgggcc gcccccgaca ccagggcgtc 240 atggtcggta tgggtcagaa agattcctac gtgggcgacg aggctcagag caagagaggt 300 atcctgaccc tgaagtaccc tatcgagcac ggcatcatca ccaactggga tgacatggag 360 aagatctggc accacacctt ctacaacgag cttcgcgtgg ctcccgagga gcaccccacc 420 ctgctcaccg aggcccccct caatcccaag gccaaccgcg agaagatgac ccagatcatg 480 tttgagacct tcaacgtgcc cgccatgtac gtggccatcc aggccgtgct gtccctctac 540 gcctccggca ggaccaccgg catcgtgctg gactccggcg acggcgtcac ccacaacgtg 600 cccatttatg agggctacgc gctgccgcac gccatcatgc gcctggacct ggcgggccgc 660 gatctcaccg actacctgat gaagatcctc actgagcgtg gctactcctt cgtgaccaca 720 gctgagcgcg agatcgtgcg cgacatcaag gagaagctgt gctacgtggc cctggacttc 780 gagaacgaga tggcgacggc cgcctcctcc tcctccctgg aaaagagcta cgagctgcca 840 gacgggcagg tcatcaccat cggcaacgag cgcttccgct gcccggagac gctcttccag 900 ccctccttca tcggtatgga gtcggcgggc attcacgaga ccacctacaa cagcatcatg 960 aagtgtgaca tcgacatcag gaaggacctg tatgccaaca acgtcatgtc ggggggcacc 1020 acgatgtacc ctgggatcgc tgaccgcatg cagaaagaga tcaccgcgct ggcacccagc 1080 accatgaaga tcaagatcat cgccccgccg gagcgcaaat actcggtgtg gatcggcggc 1140 tccatcctgg cctcgctgtc caccttccag cagatgtgga tcaccaagca ggagtacgac 1200 gaggccggcc cttccatcgt ccaccgcaaa tgcttctaga cacactccac ctccagcacg 1260 cgacttctca ggacgacgaa tcttctcaat gggggggcgg ctgagctcca gccaccccgc 1320 agtcactttc tttgtaacaa cttccgttgc tgccatcgta aactgacaca gtgtttataa 1380 cgtgtacata cattaactta ttacctcatt ttgttatttt tcgaaacaaa gccctgtgga 1440 agaaaatgga aaacttgaag aagcattaaa gtcattctgt taagctgcgt aaaaaaaaaa 1500 aaaaaaaaa 1509 <210> 802 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 802 gcagcagcag cagcagcag 19 <210> 803 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 803 gcagcagcag cagcagcag 19 <210> 804 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 804 agcagcagca gcagcagcag 20 <210> 805 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 805 gcagcagcag cagcagca 18 <210> 806 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 806 agcagcagca gcagcagca 19 <210> 807 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 807 agcagcagca gcagca 16 <210> 808 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 808 ctcccgacaa gctcca 16 <210> 809 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 809 tcccgacaag ctcc 14 <210> 810 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 810 gcttgcacgt gtggct 16 <210> 811 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 811 cttgcacgtg tggc 14 <210> 812 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 812 ggttgtgaac tggcag 16 <210> 813 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 813 gttgtgaact ggca 14 <210> 814 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 814 gagcggttgt gaactg 16 <210> 815 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 815 agcggttgtg aact 14 <210> 816 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 816 gctgccttcc caggcc 16 <210> 817 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 817 ctgccttccc aggc 14 <210> 818 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 818 gcactttgcg aaccaa 16 <210> 819 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 819 cactttgcga acca 14 <210> 820 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 820 gaaagctttg cacttt 16 <210> 821 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 821 aaagctttgc actt 14 <210> 822 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 822 cggaggacga ggtcaa 16 <210> 823 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 823 ggaggacgag gtca 14 <210> 824 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 824 agcctgtcag cgagtc 16 <210> 825 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 825 gcctgtcagc gagt 14 <210> 826 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 826 tcctgtagcc tgtcag 16 <210> 827 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 827 cctgtagcct gtca 14 <210> 828 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 828 gaagcgaggc ttcact 16 <210> 829 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 829 aagcgaggct tcac 14 <210> 830 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 830 acctgcccgt ctggca 16 <210> 831 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 831 cctgcccgtc tggc 14 <210> 832 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 832 ggtcagcgat cccagg 16 <210> 833 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 833 gtcagcgatc ccag 14 <210> 834 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 834 attttcttcc acaggg 16 <210> 835 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 835 ttttcttcca cagg 14 <210> 836 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 836 gaatgacttt aatgct 16 <210> 837 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 837 aatgacttta atgc 14

Claims (1)

CLAIMS 1. A method of treating an animal having type 1 muscle tension dystrophy,
a. Identifying an animal having type 1 muscle tension dystrophy; And
b. Comprising administering to said animal a therapeutically effective amount of a compound comprising a modified oligonucleotide of 10 to 30 linked nucleoside lengths targeting DMPK,
Wherein said animal having said type 1 muscular tonic dystrophy is treated.